Report - Alabama Water Resources Research Institute

FISHERY INFORMATION MANAGEMENT SYSTEMS
P.O. Box 3607 Auburn, Alabama 36831-3607
334•887•8860 FAX 334•887•6141 1•800• 659•8160
February 6, 1993
Water Resources Institute
Samford Hall
Auburn University, AL 36849
Dear Dennis Block,
Although you probably thought you would never see the day, here is the final version of the
VItellogenesis study along with the original photographs. I have made some changes to the original
text that I gave you in November.
Again, thank you for your patience. Also I would like to thank the Water Resources Institute
for providing funds to pursue what I believe was meaningful research.
Since~ely
Thomas M. Steeger
Vitellogenesis: An Assay for Determining
the Effects of Environmental Pollutants
in Fish
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Final Project Report
for
State Water Resources Research Institute Program
under Section 104, Water Resources Research Act of 1984
to
Water Resources Research Institute
Auburn University
Alabama
VITELWGENESIS: AN ASSAY FOR DETERMINING THE EFFECTS OF
ENVIRONMENTAL POLLUTANTS IN FISH
by
John M. Grizzle
Professor
Department of Fisheries and Allied Aquacultures
College of Agriculture
Auburn University
Telephone (334) 844-3474
James T. Bradley
Professor
Department of Zoology and Wildlife Science
College of Sciences and Mathematics
Auburn University
Telephone (334) 844-9262
John A. Plumb
Professor
Department of Fisheries and Allied Aquacultures
College of Agriculture
Auburn University
Telephone (334) 844-9215
1 DECEMBER 1995
Start: 06/92
End : 05/95
Project Number: 03
Title: Vitellogenesis: An Assay for Determining the Effects of Environmental
Pollutants in Fish
Investigators: Grizzle, John M., Bradley, James T. and Plumb, John A., Auburn
University, Auburn, Alabama.
Focus Categories: TS SW WQL
Congressional District: Third
Descriptors: Bioaccumulation, Bioassay, Bioindicators, Fish Eggs, Polyaromatic
hydrocarbons, Water pollution effect
ii
Table of Contents
Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sampling Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Induction of Vitellogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Blood Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Hepatic Vitellogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Hepatocyte Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Radiolabelling and Aroclor 1254 Exposures . . . . . . . . . . . . . . . . . . . . . . . . . 8
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) . . . . . . . . . . . . . 9
Vitellogenin Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Purification of Vitellogenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Receptor Mediated Uptake of Vitellogenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Protein Radio-iodination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Oocyte Isolation and Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Field Collection of Channel Catfish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Isolation of Channel Catfish Oocyte Vitellogenin Receptor . . . . . . . . . . . . . . . . . . . . 17
Membrane Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Membrane Extraction . . _. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Ligand blotting (SDS-PAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
17Jj-estradiol Induction of Additional Fishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Preparation of Polyclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Channel Catfish Hepatocyte Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aroclor 1254 Exposures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Receptor Mediated Uptake of Vitellogenin . . . . . . . . . . . . . . . . . . . . . . . . . . .
Radio-iodination of Vitellogenin . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aroclor 1254 Affects on Channel Catfish Oocyte Uptake of Vitellogenin . . .
Aroclor 1260 Residues and Vitellogenic Capabilities in Field Samples . . . . . . . . . .
Isolation and Characterization of Channel Catfish Oocyte Vitellogenin Receptor . . . .
Interspecies Comparisons of 17Jj-estradiol-induced Serum Proteins Reactivity to MonoPolyclonal Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
and
. . .
22
22
23
31
31
35
40
47
47
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
iii
Executive Summary
In vitro synthesis of vitellogenin by hepatocytes was inhibited by Aroclor 1254
at concentrations of 111 and 260 J.tg/mL. Viability of cells after 48 hours averaged
45.6% + 5.7; there was no difference {a=0.05) in viability between untreated and
Aroclor-treated hepatocytes at 48 hours. Western blots and autoradiography revealed
diminished vitellogenin in Aroclor-treated cultures compared to their untreated
counterparts. ELISA assays for vitellogenin confirmed that there was significantly
less vitellogenin in cultures treated with Aroclor concentrations of 111 and 260
J.tg/mL. There were no significant differences between vitellogenin levels in
hepatocytes receiving no chemical treatment and those receiving acetone only.
Regression analyses of vitellogenin concentration over increasing concentration of
Aroclor revealed significant slopes; regression coefficients averaged 0.884 + 0.003
(mean + standard error). Total protein assays on each of the tissue cultures revealed
no significant difference between treatment groups and failed to resolve whether
Aroclor was directly affecting vitellogenesis or whether hepatic protein synthesis in
general was being inhibited.
Studies of hepatic vitellogenesis demonstrate the usefulness of in vitro
techniques to assay effects of Aroclor 1254 on the synthesis of vitellogenin. The
ability to detect hepatic vitellogenesis in vitro and its sensitivity to Aroclor 1254
provide an assay with which to screen other environmental pollutants. This technique
affords a means of predicting the extent to which chemical residues affect hepatic
function and reproduction in native fishes.
Uptake of vitellogenin by channel catfish oocytes was measured by incubating
follicles with vitellogenin labeled with radioactive iodine 25I). A total of three
experiments, each containing 3 or 4 replicates, were conducted with Aroclor. The
first experiment had uptake values consistent with preliminary experiments involving
oocytes, i.e. 8.07% ± 1.42. In the remaining two studies, uptake was an order of
magnitude lower with values of 0.19% + 0.05 and 0.55% + 0.24, respectively. In
each of the three experiments there was a trend toward increased uptake of
vitellogenin with increasing concentrations of Aroclor 1254. In experiments 1 and 2
there were no significant differences between treatments; however, experiment 3 did
show significant differences between treatments. Oocytes treated with Aroclor at 56
J.tg/mL exhibited a higher percent of 125I in the oocyte homogenate. Incubations were
also conducted with a combination of suramin and increasing concentrations of
Aroclor 1254. Regression analysis of percent binding in suramin treated oocytes over
increasing concentrations of Aroclor showed a significant positive slope. Since
sodium suramin blocks receptor mediated uptake of vitellogenin, results from
suramin-treated oocytes suggest that the observed increase in uptake with increasing
concentrations of Aroclor is a result of some other mechanism than receptor-mediated
uptake.
Channel catfish collected by the Alabama Department of Environmental
Management from Choccolocca Creek (Logan Martin Reservoir) and Weiss Reservoir
(Alabama state line) exhibited tissue PCB residues of 34.71 ± 13.85 mg/kg {n=2)
e
iv
(mean + standard error) and 2.41 + 0.02 mg/kg (n=4), respectively. Fish tissue
residues from Weiss Reservoir represent a composite sample while PCB residues from
Logan Martin Reservoir represent the average of two separate fish. Serum
vitellogenin from these fish averaged 27.13 + 26.78 mg/mL (n=2) and 0.020
mg/mL (n=l). Polychlorinated biphenyl residues in fish collected through gillnetting Weiss Reservoir averaged 1.51 ± 0.59 mg/kg (n=7). Only one fish was
collected from Logan Martin Reservoir and it contained 7 mg PCB/kg. Fish collected
from Yates Reservoir contained no detectable PCB residues. Tissue PCB-residues in
channel catfish collected through gill-netting were significantly correlated with age of
fish (Pearson correlation coefficient = 0.47) and relative weight (Pearson Correlation
Coefficient 0.84). The gonadosomatic index of female channel catfish was positively
correlated (Pearson correlation coefficient = 0. 72) with serum vitellogenin. There
was no correlation between PCB residues and gonadosomatic indices or the dress-out
percentage of fillets. Similarly, there was no correlation between PCB residues and
serum vitellogenin levels.
Viability of primary hepatocyte cultures at 24 and 48 hours taken from fish
collected in the wild averaged 59.45 ± 7.06% and 54.27 + 7.53%, respectively. In
vitro synthesis of vitellogenin by hepatocytes was not correlated with PCB tissue
residues.
PCB residues in channel catfish fillets collected from Logan Martin and Weiss
Reservoirs were lower than the concentrations of Aroclor 1254 used in primary
cultures during Year 1 and 2 of the study; however, whole fish residues were
unknown in the wild-caught fish. In vitro culture of hepatocytes from fish collected
in Year 3 did not exhibit reduced vitellogenin capabilities. These data emphasize the
value of the in vitro vitellogenesis model for understanding the affects of chemical
pollutants.
In addition, work has been conducted on the isolation and characterization of
the vitellogenin receptor from channel catfish oocytes. The putative receptor, isolated
through extraction with octyl-,8-D-glucoside and visualized with ligand blotting, has a
molecular mass of 100 kDa. Ligand blotting using 125I-labeled vitellogenin confrrmed
that the single 100 kDa band was binding vitellogenin. Ligand blotting with
increasing levels of vitellogenin exhibited a positive dose response.
Work has also been completed on the reactivity of 17,8-estradiol-induced
proteins in serum from several species of fish to both polyclonal and monoclonal
antibodies against channel catfish vitellogenin. Both monoclonal and polyclonal
antibodies were reactive against serum from channel catfish, blue catfish (/. furcatus),
and flathead catfish (Pylodictis olivaris). Neither the polyclonal nor the monoclonal
antibodies reacted against 17,8-estradiol-induced serum from spotted gar (Lepisosteus
oculatus), common carp (Cyprinus carpio), redear sunfish (L. microlophus), and white
bass (Morone chrysops). The reactivity of the monoclonal antibody to vitellogenin of
other catfish species broadens the potential for examining the effects of environmental
pollutants on hepatic synthesis of vitellogenin.
v
Introduction
Over the past decade recreational and commercial use of aquatic resources has
increased. Along with this increase in usage has been a tendcney for Americans to
incorporate more fish into their diets. The increasing interest in fishery resources has
focused attention on pollution levels in the aquatic environment, their possible
influence on fish populations, i.e., reproduction, growth and mortality, their effect on
the marketability of commercial fishes, and on the consequences that eating
contaminiated fish has on the human population (Cordle et al. 1982; Fein et al. 1984;
Overstreet 1988). Although many pollutants entering aquatic environments culminate
in fish kills, the majority express themselved through chronic, sublethal effects.
While the presence of sublethal levels of pollutants in lake and river systems has been
established, their impact has not been well documented.
Variability of contaminate
levels in both biotic and abiotic samples has made interpretation of the results difficult
(Madenjian et al. 1994). There has remained a critical need to (1) examine how these
chemicals partition themselves in the environment, (2) to examine the phenomena of
bioaccumulation and the preferential distribution of these compounds in the organism,
and (3) to examine the effects of sublethal levels of pollutants on biological variables.
The relationship between the presence of chemical residues and their effect on
an entire ecosystem is not well understood. Studies have examined various levels of
organization from subcellular (biochemical) to whole populations. These studies have
attempted to identify "indicators" for predicting the bio-effect of chemical residues
and how these effects may translate into the overall condition of the affected
ecosystem (Hunn 1988).
One group of environmental contaminants which has become recognized as
ubiquitous is categorized under the heading of polychlorinated aromatic hydrocarbons,
most notably, polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-pdioxin. Although polychlorinated biphenyls (PCB) are no longer intentionally
manufactured, they continue to be generated as by-products of industries such as
bleached-kraft paper mills. As a result, residues of PCB in several species of fish are
orders of magnitude greater than the Food and Drug Administration's "action" level.
These residue action-levels are based on human health considerations, but levels that
harm aquatic animals remain uncertain. Assays for testing the environmental effects
of water pollutants are needed because currently available methods do not adequately
measure the consequences of high levels of residues accumulated by long-term
exposure to low concentrations of pollutants.
The acute and chronic effects of PCB have been demonstrated through the
exposure of fish to specific classes of PCB. J\roclor 1254 was selected for this study
because it represents a group of polychlorinated biphenyls (PCBs). Residues of PCBs
have been routinely identified in fish tissues collected from Alabama waters (personal
communication, Alabama Department of Environmental Management). The effects of
PCBs on fish have included skin lesions (Holm et al. 1993), skeletal deformities
(Bengtsson 1991), hepatotoxicity (Noguchi 1987), induction of the cytochrome P450dependent enzyme system in the liver (Andersson et al. 1988) and diminished
reproductive capacity (Norrgren et al. 1993; Holm et al. 1993; Ankley et al. 1991;
Bergquist 1989; Spies and Rice 1988; Mac et al. 1993; Mac and Edsall1991; Mac
2
1988; Hansen et al. 1985). One such hepatic effect that could potentially impact
reproductive success was a decrease in serum vitellogenin levels associated with in
vivo exposure of rainbow trout, Oncorhynchus mykiss, to Aroclor 1254 (Chen et al.
1985). Diminished levels of vitellogenin may directly affect the yolk available to
oviparous animals.
Egg yolk constitutes the major source of nutrients for the developing embryo
of all oviparous animals. Most of the growth of these oocytes is a result of massive
deposition of yolk proteins in a process termed vitellogenesis. Plasma vitellogenin, a
phospholipoglycoprotein, synthesized in the liver of females under the control of
estrogen, is the principal yolk protein in oviparous vertebrates (Tyler et al. 1988).
Studies on chicken, Gallus domesticus (Cutting and Roth 1973) and Xenopus laevis
(Wallace et al. 1970; Wallace and Jared 1976) demonstrated that vitellogenin is
selectively taken up from the plasma into developing oocytes. Specific cell-surface
receptors mediate the uptake of vitellogenin by the mechanism of receptor-mediated
endocytosis (Woods and Roth 1983; Tyler et al. 1988). Tyler et al. (1988) describes
the process whereby oocyte membrane-bound receptors contain specific binding sites
for the vitellogenin ligand. After its selective uptake by the oocytes, vitellogenin is
proteolytically cleaved into the yolk proteins termed lipovitellins and phosvitins.
Studies of vitellogenin uptake by fish oocytes have yielded conflicting results ranging
from fairly selective uptake (Campbell 1978; Tyler et al. 1988) to nonselective uptake
(Campbell and Jalabert 1979; Wallace 1985). Tyler et al. (1988) suggested that
nonselective uptake probably occurs as a result of adventitious engulfment during
3
vitellogenin uptake and may represent a mechanism by which chemical contaminates
gain entry into oocytes.
The primary objective for fiscal year 92-93 was to develop an in vitro assay
for determining the effects of chemical pollutants on hepatic synthesis of vitellogenin.
The assay was tested with Aroclor 1254 to determine the suitability of this model to
detect the effects of polychlorinated aromatic hydrocarbons on vitellogenesis. The
project objective for fiscal year 93-94 (FY93-94) was the development of an in :Yi1rQ
assay for determining the effects of chemical pollutants on the receptor-mediated
uptake of vitellogenin by oocytes. In the final year (FY94-95) of the study, the
project objective was to utilize the hepatic vitellogenesis bioassay developed in Year 1
and Year 2 to examine the effects of pollutants on fish collected in Alabama. The
reservoirs selected as fish collection sites were Weiss and Logan Martin Reservoirs on
the Coosa River and Yates Reservoir on the Tallapoosa River. These reservoirs were
selected because they differ in accumulated levels of chlorinated hydrocarbons and
related compounds. Furthermore the three reservoirs represent a broad spectrum of
environmental qualities, fish population densities, and reflect differences in the water
drainages which formed the reservoirs. Yates Reservoir is one of the least polluted
and has one of the lowest nutrient levels while Weiss Reservoir maintains the highest
nutrient level in Alabama's reservoirs
4
Sampling Sites
Impounded in 1961, Weiss Reservoir is located in Cherokee County, Alabama,
and represents the first impoundment of the Coosa River after its formation at the
confluence of the Etowah and Ostanauga Rivers in Rome, Georgia. Additional
tributaries to the reservoir include the Little and Chattooga Rivers. The reservoir has
719 km of shoreline and at normal pool elevation of 172 m, has a surface area of
12,222 ha with an average depth of 3.08 m. Major municipalities in the vicinity of
the reservoir include the city of Rome, Georgia, and the towns of Centre, Cedar
Bluff and Leesburg, Alabama.
Impounded in 1964, Logan Martin Reservoir is located in St. Claire and
Talladega Counties, Alabama, and represents the third impoundment of the Coosa
River. The reservoir has 442 km of shoreline and at normal pool elevation of 142 m,
has a surface area of 6, 177 ha with an average depth of 5.46 m. Choccolocco Creek
serves as a tributary to Logan Martin Reservoir. The major municipality in the
vicinity of the reservoir is Pell City, Alabama.
Impounded in 1928, Yates Reservoir is located in Elmore and Tallapoosa
Counties, Alabama, and represents the third impoundment of the Tallapoosa River.
The reservoir has 74 km of shoreline and at normal pool elevation of 105 m, has a
surface area of 809 ha with an average depth of 3. 96 m. Saugahatchee and
Chainahatchee Creeks serve as tributaries to Yates Reservoir. Tallassee is the major
municipality in the vicinity of the reservoir.
5
Methods
Induction of Vitellogenesis
One- to 2-year-old channel catfish were obtained from the Auburn University
Fisheries Research Station and maintained in 100-liter fiberglass tanks provided with
flowing well water at 18 to 22°C. Vitellogenin synthesis was induced in male
channel catfish with intraperitoneal injections of 17J3-estradiol (Sigma Chemical
Company) (2 mg/100 g body weight) dissolved in propylene glycol. Seven days
following the initial injection with estradiol the fish were given a second injection of
the same dose of 17J3-estradiol.
Blood Collection
Three days after the second injection of estradiol, fish were anesthetized with
tricaine (Argent) neutralized to pH 7 with sodium bicarbonate. Blood was withdrawn
from the caudal vein with a 20-gauge hypodermic needle attached to a 20-mL syringe
containing 20 mM of phenylmethyl-sulfonyl fluoride (PMSF), a proteolytic enzyme
inhibitor, dissolved in ethanol. Blood was placed in the refrigerator, allowed to clot,
and the serum removed after 24 hr. Serum was either used immediately for selective
precipitation of vitellogenin by EDTA with magnesium chloride (Wiley et al. 1979),
or was frozen at -70°C and used later for isolation of vitellogenin through preparative
isoelectric focusing. Vitellogenin isolated in this manner was used as a reference
standard.
6
Hepatic Vitellogenesis
Hepatocyte Isolation
Following blood collection, the anesthetized fish was killed by a sharp blow to
the head. Afterwards, the ventral isthmus was severed and any blood remaining in
the fish carcass was allowed to drain and was discarded. The catfish was dipped in a
0.05% solution of sodium hypochlorite; the outside of the fish was then swabbed with
95% ethanol and the liver removed aseptically. The liver was kept in a sterile Petri
dish on ice while it was trimmed of excess connective tissue. Sterile scissors were
used to thoroughly mince the liver, and fragments were washed three times with
phosphate-buffered saline (137 mM sodium chloride, 2. 7 mM potassium chloride, 6.5
mM disodium hydrogen phosphate, 1.5 mM potassium dihydrogen phosphate, 0.9
mM calcium chloride, 0.5 mM magnesium chloride). Following each wash the
fragments were centrifuged for 5 min at 1000 x gravity. Afterwards the washed
fragments were transferred to 25-mL digestion flasks containing 15 mL trypsin digest
mixture: 0.25% trypsin in phosphate buffered saline containing 2.5% fetal bovine
serum (ICN), penicillin and streptomycin (Sigma) 200 IU/mL and 200
respectively, Gentamycin at 50
~g/mL,
~g/mL,
and a magnetic stir bar. The tissue was
stirred in sealed incubation flasks at 22°C for 4 hr. The mixture of isolated
hepatocytes and nondigested liver was allowed to settle and the harvested hepatocytes
(supernate) were centrifuged at 1000 x gravity for 5 min. Hepatocytes were washed 3
times in phosphate buffered saline by centrifuging cells at 1000 x gravity for 5 min
each time and resuspending pellet in fresh phosphate buffered saline. After the final
7
wash, cells were resuspended in minimum essential medium (Gibco) with 0.33%
HEPES, 20% fetal bovine serum and penicillin and streptomycin (200 IU/mL and 200
1-'g/mL, respectively). Hepatocyte concentration was determined with a cell
cytometer. The cell suspension volume was adjusted by addition of medium to yield
approximately 30 X lOS cells/mL, and 1-mL aliquots of the cell suspension were then
pipetted into each 2-mL culture well of 24-well plastic culture plates (Coming).
Differences between viability in each of the treated and untreated hepatocyte cultures
were determined by exclusion of 0.4% (w/v) trypan blue in phosphate-buffered saline.
Hepatocyte cultures were incubated at 20°C for 48 hr.
Radiolabelling and Aroclor 1254 Exposures
Primary cultures of channel catfish hepatocytes isolated from three male
channel catfish averaging 821 grams, induced in vivo with 17/j-estradiol, were
incubated with 20 J.'Ci 33P-orthophosphate (Dupont) 8 hr after the addition of Aroclor
1254 (see below). Phosphorous-33 was selected for use in labeling because of its
preferential incorporation into the 150 kDa-vitellogenin protein. Additionally, the
extended half-life of 33P (22.4 days) compared to 32P (12 d) provided sufficient time in
which to conduct studies while assuring that the overall half-life of radioactive PCBcontaminated waste generated in experiments with Aroclor would not be excessive.
The Aroclor working dilution was prepared by heating the stock solution
(Mansanto Lot# KBOl-64) in a water bath at 100°C and delivering 10 J.'L (39.84 mg)
into a vial. The Aroclor was then dissolved in 1 mL of HPLC-grade acetone
(Mallenckrodt). Aroclor was added to individual wells within 5 min after 1 mL of
8
cell suspension had been added. Stock solutions of Aroclor 1254 ranged in
concentration from 1. 8 to 52 p.g/ p.L in acetone; 5 p.L of the stock solutions was added
to each well to yield final concentrations of Aroclor ranging from 9 to 260 mg/L.
Control cells were exposed either to 5 p.L acetone or received no treatment. Fortyeight hours after the addition of Aroclor 1254, 0.1 mL of 10 mM sodium fluoride,
10mM EDTA, and 10 mM PMSF were added to each cell culture well, and the
contents of the well were sonicated for 5 seconds. An aliquot (0.1 mL) from each
well was taken for electrophoresis, Western blot, and autoradiography. Duplicate
aliquots (0.2 mL) were taken for enzyme linked immuno-absorbant assay (ELISA),
following the procedure outlined by Goodwin et al. (1992), to determine total
vitellogenin. Plates were read with a Perkin-Elmer Lamda reader with automatic
background subtract. Additionally, an aliquot from each of the culture wells was
taken for total protein analysis with a BioRadQI> protein assay kit.
Radiolabeled-vitellogenin was detected by autoradiography of electrophoresis
gels and Western blots. Destained gels and Western blots containing radiolabeled
protein were photographed, and autoradiographs obtained on Kodak XAR-5 film were
exposed for 60 days at -70°C.
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Samples for electrophoresis were mixed with an equal volume of sample buffer
(5 mL mM Tris-base pH 6.8, 5% sodium dodecylsulfate, 5% 2-mercaptoethanol, 50%
glycerol, and bromophenol blue) heated at 100°C for 1 min, and stored at -70°C.
Molecular mass and vitellogenin standards were prepared in the same manner as the
9
experimental samples. Molecular mass standards (BioRad) consisted of myosin (205
kDa), (3-galactosidase (116.25 kDa), phosphorylase b (97.4 kDa), bovine serum
albumin (66.2 kDa), and ovalbumin (42.7 kDa). Electrophoresis was conducted by
applying 60 p.L of experimental samples and 20 p.L of molecular mass markers into
separate wells of two 20-well, 1.5 mm, 7.5% polyacrylamide gels and run overnight
at 35 volts at 22°C until the bromophenol blue marker migrated to within 1 em of the
gel bottom (approximately 24 hr duration). For each set of samples, one gel was
stained for total protein with Coomassie blue R, and a second gel was assayed for
vitellogenin by Western blot.
Vitellogenin Assays
Vitellogenin was assayed using both Western blot and enzyme-linked
immunoabsorbent assay. Immediately after SDS-PAGE, polypeptides were
electroblotted onto nitrocellulose membrane (BioRad) with a BioRad trans-blot cell.
Before transfer, the gel was soaked 30 min in 4 M urea in transfer buffer (0.025 M
Tris base, 0.11 M glycine, 20% methanol). Electroblotting was performed for 1 hr at
1 amp. Protein bands were visualized with 2% Ponceau S stain; the location of
molecular mass markers was noted by indenting the nitrocellulose. The blot was
rinsed in Tris-buffered saline (TBST; 20 mM Tris-base and 500 mM NaCl, pH 7.5,
containing 0.05% Tween 20), blocked with 2.5% milk in TBST for 1 hr, and then
incubated overnight at 5°C with a 1: 1, 000 dilution of monoclonal antivitellogenin
antibody produced against estradiol-induced plasma vitellogenin produced in male
channel catfish (Goodwin et al. 1992) in 2.5% milk in TBST. After two 10-min
10
washings with TBST the blot was transferred to 50 mL of 2.5% milk in TBST
containing 1:20,000 goat anti-mouse IgG alkaline phosphatase conjugate (Sigma
Chemical Co.) and incubated overnight at 5°C. After another two 10-min washings
with TBST, 10 mL nitro blue tetrazolium (10 mg NBT/50 mL 100 mM Tris-HCL)
and 100 p.L of 5-bromo-4-chloro-3-indolyl phosphate (25 mg BCIP/1.25 mL dimethyl
sulfoxide) were added and the reaction allowed to proceed at 22°C until color
development within 5 min.
Purification of Vitellogenin
Vitellogenin used for ligand binding was obtained by preparative isoelectric focusing
(IEF) using the BioRad Rotofor cell. The IEF chamber was pre-run using tissue
culture-grade distilled water until a reading of 2.0 rnA was obtained. The chamber
was then prefocused for 30 min using 2% v/v BioLytes (pH range 4 - 6.5). Focusing
was conducted at 12 watts constant power using a BioRad 3000xi power supply.
Serum (1 mL) collected from a estradiol-induced male channel catfish, was introduced
into the side of the IEF chamber closest to the anode. After 4.5 h the chamber
contents were drained under vacuum, their pH measured, and 0.2 mL aliquots were
taken for SDS-PAGE. Vitellogenin was visualized by Western blotting. Rotofor
chambers containing the 150 kDa vitellogenin band were then lyophilized
Receptor Mediated Uptake of Vitellogenin
Protein Radio-iodination
Iodine-125 was selected for labeling channel catfish vitellogenin because of the
relative ease of the labeling procedure, the high specific activity of the labeled
11
protein, and the reasonable half-life (60 days) of the label. Because of the hazards
associated with PCBs, Aroclor 1254, a short-lived radioisotope was used to eliminate
the need of disposing of radioactive polychlorinated biphenyls.
Vitellogenin, purified as described above, was dissolved in 0.05 M phosphate
buffered saline to yield a 1 Jlg/ Ji.L solution. To 7 Ji.L vitellogenin 1 mCi 125-lodide
was added followed by 25 Ji.L chloramine-T (12 mg/10 mL) and the reaction vial was
gently mixed. Forty-five seconds after the addition of chloramine-T, 25 Ji.L of
sodium metabisulfite (28.5 mg/10 mL) was added.
vitellogenin labeled with
The reaction mixture, containing
125
1, was repurified with gel filtration (Sephadex G-200).
Equilibration of the gel and elution of vitellogenin was carried out with 0.5 M
phosphate buffer saline, pH 7.4, containing 1% bovine serum albumin. Equilibration
of the gel with albumin was used to 'presaturate' the Sephadex column to increase
recovery of 1251-labeled protein (Greenwood et al. 1963). The eluted fractions with
the highest activities were applied to duplicate 12% polyacrylamide mini-gels. One
gel was used for Western blot and the second gel was stained with Coomassie blueR
for proteins. The stained gel and the blot were dried and used for autoradiography.
Stock
125
1-vitellogenin was stored at -70°C; at no time did the temperature of the stock
solution exceed 10°C.
Oocyte Isolation and Exposure
Two- to three-year-old channel catfish were anesthetized with tricaine. The
catfish was dipped in a 0.05% solution of sodium hypochlorite (bleach); the outside of
the fish was then swabbed with 95% ethanol and the ovary removed aseptically. The
12
ovary was kept in a sterile Petri dish on ice while it was trimmed of excess
connective tissue. Sterile scissors were use to remove sections of the ovary which
could be used for isolating individual follicles. Ovary sections were washed a
minimum of three times with phosphate-buffered saline (PBS) (137 mM sodium
chloride, 2. 7 mM potassium chloride, 6.5 mM disodium hydrogen phosphate, 1.5
mM potassium dihydrogen phosphate, 0.9 mM calcium chloride, 0.5 mM magnesium
chloride). Each wash was decanted from the petri dish and discarded. Outer layers
of the follicles were removed manually with iridectomy scissors. Isolated oocytes,
divested of outer layers, were then transferred to 24-well plastic culture plates
(Coming) containing minimum essential medium (GIBCO) with 0.33% HEPES, 20%
fetal bovine serum and penicillin and streptomycin (200 IU/mL and 200 1-'g/mL,
respectively). Oocytes were examined under a light microscope and the viability of
oocytes determined using the method developed by Tyler et al. (1990). Oocytes
exhibiting uniform yolk were determined to be viable while oocytes having yolk
coalesced in a 'halo' were termed 'nonviable' and were removed from the culture
medium. While this technique was applicable to cells readily viewed under a light
microscope, i.e. cell < 1 mm diameter, oocytes with a diameter
no way of determining their viability.
> 1 mm afforded
After a 4-h incubation, oocytes were washed 3
times with 1.0 mL of 10 mM sodium fluoride, 10 mM EDTA, and 1 mM
phenylmethyl sulfonyl fluoride; washes were combined with their respective
incubation medium. Oocytes were sonicated with 0.5 mL of 10 mM NaF, 10 mM
EDTA, and 1 mM PMSF. Duplicate 20 J.'L aliquots of the oocyte wash and
13
homogenate were taken for counting. Uptake of 125I-vitellogenin was determined by
dividing the total radioactivity in the homogenate by the total radioactivity in the
medium and oocyte homogenate; the quotient was then expressed as a percent. In
incubations with Aroclor, uptake of 125I-vitellogenin was determined by the percent of
counts present in the homogenate minus the percent of nonspecific binding determined
from sodium suramin treated incubations (see below).
Oocytes, 2 mm in diameter, were incubated with Aroclor 1254 for 4 hours.
Similar to the hepatocyte exposures, 5 J.LL of Aroclor stock solution was added to each
culture well containing 10 oocytes in 1 mL of medium to yield final concentrations of
Aroclor ranging from 9 to 260 J.LglmL (ppm). Controls cells were exposed either to 5
J.LL acetone or received no treatment.
Sodium suramin (1, 3, 5 naphthalenetrisulfonic acid 8, 8' [carbonyl bis [imino3,1-phenylenecarbonylimino (4-methyl-3, 1-phenylene (carbonyl-imino]]-bishexasodium salt) (C. B. Chemicals, Inc.) was used to inhibit uptake of vitellogenin
and document the level of nonspecific binding of 125I-vitellogenin. Working solutions
were prepared by dissolving 1 g suramin in 10 mL PBS. To determine whether
Aroclor was affecting receptor-mediated uptake oocytes, treated with sodium suramin,
were incubated with concentrations of Aroclor ranging from 9 to 260 mg/L.
Field Collection of Channel CatiiSb
Vitellogenic capabilities were assessed for fish collected from Weiss and
Logan Martin Reservoirs on the Coosa River and Yates Reservoir on the Tallapoosa
River. Fish were initially collected from Weiss and Logan Martin Reservoirs in
14
conjunction with the Alabama Department of Environmental Management through
both electrofishing and gill netting from October through November 1994; however,
only blood samples were obtained from these fish. Additional fish were subsequently
collected through experimental gill netting conducted on the three reservoirs from
February- through June 1995.
Experimental monofilament nylon gill nets consisted of five 15-m mesh-size
panels: 2.5, 3.8, 5.1, 7.6, 10.2 em bar-mesh. A total of 5 nets were set for a period
of 24 hours (net night) per sample period. Live channel catfish were harvested from
the gill nets and transferred to live wells for transport back to Auburn University.
Fish were maintained in 3.7 x 1 x 0.6 m (1,779 1) cement holding tanks. Fish were
anesthetized and then bled from the caudal vein. Fish were weighed (g) and
measured for total length (TL) then sacrificed and their livers removed for isolation of
hepatocytes. Relative weights (Wr), the weight of individual fish divided by a
standard weight for fish of that length, were calculated. Additionally, ovaries, trunk
kidney and spleen were removed and weighed. Organ-somatic indices were calculated
by dividing the organ weight by the total body wieght; indicies included the
gonadosomtic (GSI), renalsomatic (KSI) and splenosomatic (SSI). Fish were frozen
after removal of internal organs.
Fish were filleted on a cutting board covered with heavy duty aluminum foil
that was changed between fish. Care was taken to avoid contaminating fillet tissue
with material released from the inadvertant puncture of internal organs. Fish were
filleted while ice crystals were still present in the muscle tissue. A clean, stainless
15
steel fillet knife was used to remove both fillets from each fish. Fillets were obtained
by making a shallow cut through the skin (on either side of the dorsal fish) from the
top of the head to the face of the caudal fin. A second cut was then made behind the
entire length of the opercular flap, cutting through the skin and flesh to the bone. A
third shallow cut was made along the ventral surface of the fish from the base of the
pectoral fin to the anal pore. Both fillets were then removed from the fish and
weighed and the wieghts recorded to the nearest gram. Fillets were then wrapped in
heavey duty aluminum foil and labeled with the sample identification number, the
sample type (e.g. fillet) and the date of resection. Samples were then stored at -20°C
until analysis. Dressout percentage was calculated by dividing the weight of fillet by
the total body weight.
Primary culture of channel catfish hepatocytes was maintained in Eagles'
minimum essential medium with 20% fetal bovine serum and penicillin/ streptomycin
following the procedure outlined above. Hepatic synthesis of vitellogenin in vitro at
24 and 48 hr was determined by enzyme linked immunoabsorbant assay (ELISA).
Chemical residue analysis of skinless fillets and ovaries were performed by the
Alabama Department of Agriculture and Industries, Pesticide Residue Laboratory
Division. Residue analysis followed the procedure for small scale extraction of fat
with sodium sulfate and petroleum ether in the Pesticide Analytical Manual (U. S.
FDA 1990). Vitellogenin levels in serum and primary culture media were determined
through ELISA and were then compared to the concentration of pollutants (Aroclor
1260) in fillets.
16
Age of fish was determined by sectioning pectoral spines. Cross-sections of
pectoral spines were affixed to glass slides using thermal resin; the section was then
ground to a thickness of 1 mm using a Wen wet stone (Model #2900 Type 1).
Sectioned spines were examined through a dissecting microscope for annular marks.
Distance from the kemal to each annular mark was measured using an ocular
micrometer.
Age was calculated using the direct ratio method.
Isolation of Channel Catnsh Oocyte Vitellogenin Receptor
Membrane Preparation
Two- to three-year-old female channel catfish were anesthetized using tricaine
(Argent Chemical Co.). Ovaries were removed and placed in a sterile Petri dish on
ice. All procedures were then carried out at 4°C. Channel catfish ovary was
homogenized in 10 mL of membrane preparation buffer (20 mM Trizma
hydrochloride, 2 mM calcium chloride, 150 mM sodium chloride, 1 mM phenyl
methyl sulfonyl fluoride [PMSF], 2 JLM leupeptin) per gram of tissue with a Polytron
homogenizer (30 sec at setting 5 followed by two periods of 20 sec each at setting 8).
Large debris was removed by centrifugation at 5,000 g for 5 min. The supernate was
then transferred to clean tubes andre-centrifuged at 5,000 g for 5 min. Supernate
was pipetted into 13 x 51 mm Beckman Polyallomer centrifuge tubes and centrifuged
at 100,000 g for 1 h. Supernate was discarded and pellets resuspended in membrane
preparation buffer by aspiration through a 22-gauge needle and re-sedimented by
centrifugation at 100,000 g for 1 h. The procedure was repeated once. The washed
17
membrane pellet was resuspended in buffer, transferred to cryotubes and stored in
liquid nitrogen or extracted immediately.
Membrane Extraction
Membrane preparations in buffer were re-sedimented by centrifugation at
100,000 g for 1 h. Supemates were discarded and the pellet re-suspended by
aspiration through a 22-gauge needle in 1. 0 mL buffer containing 250 mM
Tris/maleate (pH 6.0), 2 mM CaC12 , 1 mM PMSF, and 2 p.M leupeptin.
Suspensions were sonicated for 20 seconds 2 times. Protein concentrations in each of
the suspensions was determined using BioRad® protein assay kit. Samples were then
centrifuged at 100,000 g for 1 hr tore-sediment the pellet. Supernates were
discarded and pellets were re-suspended in sufficient volumes of membrane extract
buffer (125 mM Tris-maleate [pH 6.0], 2 mM CaC12 , 160 mM NaCl, 1 mM PMSF, 2
JLM leupeptin, 36 mM octyl-{j-D-glucopyranoside) to yield protein concentrations
between 1 - 5 mg/mL. Suspensions were stirred for 10 min. Afterwards
undissolved material was removed by centrifugation at 100,000 g for 1 h. Cleared
supernate was adjusted to 50% acetone (acetone at -20°C) and then centrifuged at
100,000 g for 30 min. Pellets were then re-suspended by aspiration through a 22gauge needle in 25 mM Tris/HCI (pH 8.0), 50 mM NaCl, and 2 mM CaC12 •
Ligand blotting (SDS-PAGE)
For ligand blotting experiments, samples were not reduced and not heated
before loading to polyacrylamide gels. Aliquots (0.1 mL) from each sample to be
electrophoresed were treated with 0.1 mL of sample buffer (5 mL stacking gel buffer,
18
50% glycerol, 10% SDS, bromphenol blue). Molecular mass and vitellogenin
standards were prepared by treating standards with an equal volume of sample buffer
containing sodium dodecylsulfafe, 2-mercaptoethanol, glycerol, and bromphenol blue,
heat treated at 100°C for 1 min and stored frozen until electrophoresis.
Electrophoresis was conducted by applying 20 p.L of receptor prep and 15 p.L of
molecular mass markers into separate wells of lO-well, 0.75 mm, 7.5%
polyacrylamide minigels and run at 30 rnA at 5°C until the bromphenol blue marker
migrated to within 0.5 em of the gel bottom (approximately 2.5 hours duration).
Immediately after SDS-PAGE, polypeptides were electroblotted onto
nitrocellulose membrane (BioRad) using a BioRad trans-blot cell. Electroblotting was
performed for 1.5 hat 275 rnA. Protein bands were visualized using 2% Ponceau S
stain. The location of molecular mass markers was noted by indenting the
nitrocellulose. The blot was rinsed in ligand blotting buffer (LBB) containing 25mM
Tris/HCl (pH 8.0), 50 mM NaCl, and 2 mM CaC12 • The blot was blocked with 5%
milk-LBB for 30 min at room temperature and then overnight at 5°C. Ligand
incubations, using both unlabeled and
125
1-labeled channel catfish vitellogenin, were
conducted for 30 min at room temperature and then overnight at 5°C. Ligand
incubations were also conducted using 17,8-estradiol-induced flathead catfish serum.
The blot was rinsed for 2, ten-minute periods with LBB and then incubated overnight
at 5°C with a 1:1,000 dilution of antivitellogenin antibody in 5% milk in LBB
containing 0.05% Tween 20 (LBBT). After 2 ten-minute washings with LBBT the
blot was transferred to 50 mL of 5% milk in LBBT containing 1:20,000 goat anti-
19
mouse IgG alkaline phosphatase conjugate (Sigma Chemical Co.) and incubated
overnight at 5°C. After another 2 ten-minute washings with LBBT, 10 mL NBT and
100 ~-tL BCIP were added and the reaction allowed to proceed at 22°C until color
development within 5 min. On incubations utilizing
125
1-labelled vitellogenin,
autoradiograms were obtained by exposing the dried nitrocellulose paper to Kodak XOMAT film at -70°C.
17P-estradiol Induction of Additional Fishes
Spotted gar (Lepisosteus oculatus), blue catfish (lctalurus furcatus), white
catfish (Ameiurus catus), flathead catfish (fylodictis olivaris), yellow bullhead
(Ameiurus natalis), common carp (Cyprinus carpio), redear sunfish a&wmis
microlophus), and white bass (Morone chrysops) were collected with electrofishing in
Yates Reservoir, Tallapoossa County, Alabama. Fish were collected using a SmithRoot 5 GPP electrofisher at 6 amps and 120 pulses per second. Wisconsin-style
umbrella anode arrays extended from the bow of a Smith-Root workboat; the boat
hull served as the cathode. Fish were transported back to the same holding facility
used for channel catfish. Blood samples were drawn from the caudal vein; afterwards
the fish were induced with 17{j-estradiol following the procedure outlined for channel
catfish.
Preparation of Polyclonal Antibodies
Purified channel catfish vitellogenin (1 mg), obtained with the BioRad
Preparative IEF chamber (see above) and dissolved in 0.5 mL PBS, was combined
with 0.5 mL complete Freund's adjuvant (#12N9021). The mixture was sonicated at
20
4°C in a 3-mL plastic syringe until the emulsion had a thick consistency where a drop
of the emulsion when placed on water would not disperse. The entire 1-mL content
of the syringe was injected sub-cutaneously at 4 different sites in the nape of a male
New Zealand White rabbit. Four weeks later the rabbit was again injected (boosted)
with 1 mg antigen emulsified in incomplete Freund's adjuvant (1:1). A repeat booster
immunization was given 2 weeks after the initial boost. Ten days after the second
booster immunization, the rabbit was restrained and 5 mL of blood collected from the
marginal ear vein. Specific antibody titer of the antiserum was determined by
ELISA.
Western blots using the polyclonal antibodies were conducted similar to the
Western blotting procedure described above with the following modifications.
Primary antiserum was first absorbed by incubation a 1:1,000 dilution of the
polyclonal antibody with 2.5% milk in Tris-buffered saline, 0.05% Tween-20, and
nitrocellulose paper. Afterwards, the nitrocellulose was removed and the trans-blot
was added. A second modification involved the use of alkaline phosphase-conjugated
goat anti-rabbit IgG as the secondary antibody.
Statistical Analyses
Differences between treatment groups in cell viability and in vitellogenin
concentration were tested with an analysis of variance in the general linear models
(GLM) procedure of SAS (SAS Institute, Inc., 1989); multiple means comparisons
were conducted with Tukey's Studentized range test (a=0.05). Vitellogenin
21
concentration was regressed on concentration of Aroclor 1254. The regression
coefficient was considered significant at a=0.05.
Differences between treatment groups in uptake of 125I-vitellogenin by oocytes
were examined using an analysis of variance in the General Linear Models (GLM)
procedure of SAS (SAS Institute, Inc., 1989); multiple means comparisons were
conducted using Tukey's Studentized Range Test (a=0.05).
Correlation analyses were performed using the correlation procedure of SAS
(SAS Institute, Inc., 1989) Pearson Correlation coefficients were considered
significant at a=0.05.
Results
Channel Catf"lsh Hepatocyte Culture
Primary cultures of channel catfish hepatocytes were successfully maintained
for 48 hrs. Average hepatocyte density was 30 x 105 cells per well; larger numbers
of cells reduced viable-cell longevity. Viability of the original hepatocyte suspension
ranged from 50 to 99%, and viability after 48 hrs ranged from 10 to 50%. The
lowest viability (10%) occurred in wells with greater than 24 x 1Q4 hepatocytes per
well. In addition to hepatocytes, other cell types were also present; however, no
effort was made to quantitate their number.
Vitellogenin in 48-hr-old primary cultures of channel catfish hepatocytes was
identified immunoelectrophoretically (Figure 1). Coomassie blue staining of
electrophoresed proteins from 48-hr cell culture homogenates (Figure 2) revealed a
major 150-kDa band in each of the lanes containing hepatocyte cultures. Additional
22
bands, representing degradation of the 150 kDa-protein, were also present. Because
the 150-kDa band was the predominate band for vitellogenin and was well separated
from nonvitellogenic proteins, it was selected as the reference band for determining
the presence of vitellogenin in hepatocyte cultures.
In vitro synthesis of vitellogenin by cultured hepatocytes was verified by
incorporation of 35S-methionine and 33P-orthophosphate into the 150 kDa polypeptide
(Figure 3). While 35S-methionine was associated with a number of bands including
nonvitellogenic proteins and lower molecular weight fragments of vitellogenin as well
as the 150-kDa band, the 33P-orthophosphate appeared to be incorporated exclusively
into the 150-kDa band.
Aroclor 1254 Exposures
Cell viabilities at 48 hr ranged from 10 to 80%; average cell viability was
47%
+ 4.5 (mean + standard error). In two of the three experiments, there were no
significant differences in viability among treatments. In an experiment where viability
averaged 80% , viability was lowest in the blank control, and viability was highest for
cells in 56 p.g Aroclor/mL.
23
·kDa ...
VS~
..,._,_,.
~....__.......,
____ ..
t
Figure 1 Western blot of SDS-Page gell containing homogenates of channel catfish
hepatocytes 48 hrs after primary culture. Molecular mass markers were visualized
with Ponceau S prior to Western blotting and used to identify vitellogenin standard
(150 kDa) band (arrow).
24
2 3 4 5 6 7 8 9 10 11 12 13 14 ~Pf
MM
205 ..
as~
~· 1 h~ ~•~,.....~
116 '97 .. .
•
1
4 .. •. •
•
66
no
om
a
no
Figure 2 Coomassie blueR-stained SDS_PAGE gel containing homogenates of
channel catfish hepatocytes 48 hrs after primary culture. Molecular mass markers
(mm) and 150 kDa vitellogenin (VGS) band are indicated.
25
I
1 ~ 3 4 5 6 7 8 9 10 1112 13 141516 17 18
"
~,.
..
~
... ..
..~
•
-
...,rVGS
i...
....-
-i
Figure 3 Autoradiograph of Western blot of channel catfish hepatocyte culture after a
48-hr incubation with 35S-methionine. Vitellogenin standard (150 kDa) band is
indicated (VGS).
26
Western blot of cell homogenates 48 hrs after addition of Aroclor showed that
the amount of vitellogenin decreased with increasing concentrations of Aroclor 1254
(Figure 4). Visually there was no apparent difference in the amount of
immunoreactive vitellogenin in untreated hepatocytes and that in control cells treated
with 5 fJ.L acetone. Cultures treated with 111 or 260 f.'g/mL Aroclor exhibited
Western blots that were faint. Autoradiography of SDS-PAGE gels (Figure 5)
revealed diminished levels of 33P-vitellogenin with increasing concentrations of
Aroclor 1254. Untreated and solvent-control cell suspensions had similar intensities
in autoradiographs.
Vitellogenin concentrations in two Aroclor experiments were measured by
ELISA {Table 1). There was no significant difference between cell suspensions
receiving acetone only and the blank control. Cell suspensions treated with 111
f.'g/mL had significantly lower levels of vitellogenin than hepatocytes treated with
acetone only and 9 f.'g/mL. Cell suspensions treated at 260 f.'g Aroclor had
vitellogenin levels significantly lower than controls and hepatocytes treated with 9,
18, and 56 f.'g/mL. These results are consistent with the patterns in both Western
blots and autoradiographs and confirm that vitellogenin concentrations in primary
cultures of hepatocytes treated with Aroclor 1254 were significantly lower than in
controls.
27
Table 1 Mean vitellogenin concentrations {J.tg/mL) in primary cultures of channel
catfish hepatocytes treated with increasing concentrations {J.tg/mL) of Aroclor 1254.
Means within a column with different letters are significantly different (a=0.05).
Treatment
AroclQr ~~/mL
Mean
Stug~
1
Standard
Ermr
Stud~ 2
Mean
Standard
Ermr
Untreated
74.09b
1.52
27.67c
0.02
Acetone only
71.24b
1.90
29.13C
0.41
9
76.46()Cb
2.18
29.96bc
0.21
--
28.6bc
0.30
18
--
56
70.25b
0.41
28.08bc
0.01
111
66.26•
1.11
26.93bc
0.19
260
56.448
2.42
25.7•
0.54
28
~150kDa
Figure 4 Western blot of SDS-PAGE gel containing 48-hr channel catfish
hepatocyte primary culture homogenates with and without Aroclor 1254. Vitellogenin
standard (VG) is indicated in lane 2. Lanes 3 and 4 represent replicate untreated
cultures, lanes 5 and 6 contain replicate cultures treated with 5 p.L of acetone. Lanes
7 and 8 contain replicate cultures treated with 9 p.g Aroclor/mL and lanes 9 and 10
contain replicate cultures treated with 18 p.g Aroclor/mL. Lanes 11 and 12 contain
replicate cultures treated with 56 p.g Aroclor/mL; lanes 13 and 14 contain replicate
cultures treated with 111 p.g Arolcor/mL. Lanes 15 and 16 contain replicate cultures
treated with 260 p.g Aroclor/mL.
29
150~ :.•.
--!~
·t
' I
~;. .•.f
r,
,..,'
c,..
~
Figure S Autoradiograph of proteins electroblotted from a SDS-PAGE gel to a
nitrocellulose membrane. Samples were hepatocyte primary culture homogenates,
incubated with 33P-orthophosphate, with and without Aroclor 1254. Vitellogenin
standard (150-kDa band) is indicated in lanes 2 and 19. Lane 3 represents untreated
hepatocytes, lane 5 contains hepatocytes treated with 5 p.L of acetone. Lanes 7 and 8
contain hepatocytes treated with 9 p.g Aroclor/mL and lanes 9 and 10 contain
hepatocytes treated with 18 p.g Aroclor/mL. Lanes 11 and 12 contain hepatocytes
treated with 56 p.g Aroclor/mL; lanes 13 and 14 contain hepatocytes treated with 111
p.g Arolcor/mL. Lanes 15 and 16 contain hepatocytes treated with 260 p.g
Aroclor/mL.
30
Vitellogenin concentrations per 1Q4 cells were calculated because of the
differences in cell viability between the two studies. Regression analysis of corrected
vitellogenin concentration on Aroclor concentration (Figure 6) indicated that
vitellogenin concentration decreased with increasing concentration of Aroclor
(correlation coefficients averaged 0.884
+ 0.003).
Amount of total protein in each of
the cell cultures was not significantly affected by Aroclor 1254.
Receptor Mediated Uptake of Vitellogenin
Radio-iodination of Vitellogenin
Isolation of channel catfish vitellogenin used as reference standards and
labeling with [1251]-iodide proved problematic due to the instability of the protein.
Teleost vitellogenin is less phosphorylated compared to other vertebrates (Wallace
1985; Mommsen and Walsh 1988) rendering its isolation using more traditional
techniques, eg. EDTA/Mg2 + and ion exchange chromatography, less effective.
Isoelectric focusing using the BioRad IEF Preparative chamber was effective;
however, teleost vitellogenin readily undergoes degradation (Silversand and Haux
1989; Tyler and Sumpter 1990; Goodwin et al., 1992; Silversand et al., 1993) and its
use required low temperatures and protease inhibitors from collection of the blood and
throughout its isolation (Silversand et al. 1993).
Western blot (Figure 7) and autoradiograms (Figures 8) indicated that the
major vitellogenin band {VTG), i.e. 150 kDa, was labeled with
125
1. The dark band,
corresponding with the 66-kDa standard, represents bovine serum albumin used to
equilibrate and elute the Sephadex G200 column. The 1251-vitellogenin eluted from the
31
~------------------------------------~
20 1-" ~
-
-
..
---.
- - -•- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
------t.
Ill
--- ... ----
~
(,)
0 15
~
........
c
~
00
0
~
::::
10 --------~---J
>
------ - - -•- - - - 1 .
!
--- -·
•
5
Or-----+:----~:~----+-}----~:------~:--~
0
9
18
56
111
260
300
p.g Aroclor/mL
Figure 6 Mean vitellogenin concentration (/lg/lxlO" cells/mL) in primary cultures of
channel catfish hepatocytes treated with increasing concentrations (/lg/mL) of Aroclor
1254. Data points
<• and
•) represent two separate experiments using different fish.
Dashed lines (---) represent separate regressions for each of the two experiments.
32
2
Q
3
4
6
5
........ .....
7
~..,~
~
=
----.
. .- .
9
8
-•
I
""'150
I
MM
-205kOa
-116
-97
I -66
--43
--·-29
Figure 7 Coomassie blue R stained gel of 1251-labeled vitellogenin eluates 13-15 from
Sephadex G200 column. High molecular mass markers (205, 116, 97.4, 66, 43, and
29 kDa) applied in lanes 2 and 9. Lanes 4, 5, and 6 contain 10 JLL Sephadex G200
column eluates 13, 14, and 15; lane 7 contains vitellogenin standard.
33
3
4
6
5
7
-
~
9
-205·
-
~
....
...... 150klra
~
--66
•
-43
........
Figure 8 Autoradiograph of Coomassie blueR stained gel of 1251-labeled vitellogenin
eluates #13 - #15 from Sephadex G200 column. Lanes 4, 5, and 6 contain 10 #-'L
Sephadex G200 column eluates 13, 14, and 15.
Position of high molecular mass
markers (205, 116, 97.4, 66, 43, and 29 kDa) applied in lanes 2 and 9 and a
vitellogenin standard applied to lane 7 are indicated.
34
Sephadex column relatively early, i.e. within the first 14 mL collected. A total of 3
column fractions (#13, 14,15), each consisting of 1.5 mL, contained 28
~-tCi, and 22.2 ~-tCi
~-tCi,
39.9
125
1, respectively. Assuming that all of the vitellogenin (7 J.tg)
taken for labeling was represented in the three column fractions, an estimate of the
specific activity is the total activity in the three fractions divided by the total
micrograms of vitellogenin labeled.
Specific
Activity
_ 90 . lJ..LCi _ 12 . 9 J..LCi
- 1J.,LgVI'GJ.,Lg
This calculation yields roughly 12.9 ~-tCi
125
1 per J.tg of vitellogenin. The Sephadex
G200 column was effective at separating out any free
125
1 from the labeled
vitellogenin.
Aroclor 1254 Affects on Channel Catfish Oocyte Uptake of Vitellogenin
A total of three experiments were conducted assaying the effects of Aroclor
1254 on in vitro receptor mediated uptake of vitellogenin by channel catfish oocytes.
Four-hour incubations to determine the effects of Aroclor 1254 were conducted on
oocytes 2 mm in diameter. Table 2 presents the results of each experiment; the first
experiment had an average uptake across all treatments of 8.07
+
1.42% while
experiments 2 and 3 had average uptakes across all treatments of 0.18
0.55
± 0.24%,
±
0.05% and
respectively, which were an order of magnitude lower. Experiments
2 and 3 were conducted during 2 weeks in early May while Experiment 1 was
35
conducted one month earlier. Oocytes used in the last two experiments were almost
completely developed and close to ovulation while the oocytes used for the first
experiment were not as developed.
Sodium suramin was used to establish the level of nonspecific binding.
Oocytes in medium containing 14 mM sodium suramin exhibited the lowest uptake
relative to the other treatments in each of the three studies. In each of the three
experiments there was a trend toward increased uptake of vitellogenin with increasing
concentrations of Aroclor 1254. In experiment 1 and 2 there were no significant
differences between treatment groups; however, experiment 3 showed significant
differences between treatments. Oocytes treated with 56 p.g/L Aroclor exhibited a
higher percent of 125I in the oocyte homogenate.
In order to document whether Aroclor was affecting oocyte membrane
permeability, incubations were conducted which combined suramin treatment with
increasing concentrations of Aroclor 1254. A regression analysis examining power
transformed uptake of 1251-vitellogenin over increasing concentrations of Aroclor
(Figure 9) showed that the slope was significant, i.e. oocytes whose vitellogenin
receptors had been blocked, exhibited a significant increase in uptake with increasing
concentrations of Aroclor.
36
Table 2 Uptake in percent of 125!-vitellogenin by oocytes treated with increasing
concentrations of Aroclor 1254. Corrected percent represents uptake minus percent
due to nonspecific binding as determined by sodium suramin exposure. Means with
the same letter are not significantly different (a=0.05). No significant difference for
Experiments 1 and 2.
Treatment
Suramin
Percent Uptake
(Corrected Percent)
Percent Uptake
(Corrected Percent)
Percent Uptake
(Corrected Uptake)
Exposure 1
Exposure 2
Exposure 3
11.15%
1.09%
1.77%
--
--
--
Blank
19.76%
(8.61 %)
1.23%
(0.14%)
1.95%bc
(0.18%)
Acetone
12.32%
(1.17%)
1.27%
(0.18%)
1.79%bc
(0.02%)
9 p.g/L
21.09%
(9.94%)
1.34%
(0.25%)
1.62%c
(0.00%)
18 p.g/L
16.12%
(4.97%)
1.33%
(0.24%)
1.69%bc
(0.00%)
56 p.g/L
20.09%
(8.94%)
1.01%
(0.00%)
3.27%•
(1.5%)
111 p.g/L
22.68%
(11.53%)
1.16%
(0.07%)
2.68%abc
(0.91 %)
260 p.g/L
22.47%
(11.32%)
1.51%
(0.42%)
3.00%ba
(1.23%)
37
2.5 r - - - - - - - - - - - - - - - - - - - - - - ,
c
·c:
~
~
Q
2 -- ------------------------------- --•-------•
..:It
w---.
-*""---
•f
-~
] l.S - - - - - - - - - - - - - >
'>
---~..:.,..... .....~If
II'
11'1
~
.
....
--~ ~--: _-_
-:-_ - - - - - - - - - - - - - - - - - - -
I
J 1 -------------------------------------------•
....c.=
:I
~
o.s
-- -
---------------------------------------
OL-----------------------------------------~
0
9
18
56
111
260
ug Aroclor/mL
Figure 9 Regression analysis examining power transformed uptake of 125Ivitellogenin over increasing concentrations of Aroclor in channel catfish oocytes
treated with sodium suramin. Data points (•) represent observed values; dashed line
(--*--) represents regression.
38
The use of 1251-vitellogenin to assay for receptor-mediated uptake into oocytes
in vitro was accomplished, however, the percent of uptake varied considerably
depending on the time of year. In channel catfish, vitellogenic follicles are all
approximately the same size and are subsequently all ovulated at one time. This
synchrony in follicle development means that a complete compliment of vitellogenic
follicles at different stages of development cannot be obtained from a single female.
Difficulties in obtaining purified vitellogenin slowed progress on vitellogenin uptake
studies. Uptake was examined over a 4-month period during which time the size of
the follicles ranged from 0.5 to 2 mm diameters. Follicle size has been demonstrated
to have considerable bearing on the rate of vitellogenin uptake; while the initial rate
of vitellogenin uptake is high in vitellogenic follicles, as follicles approach ovulation,
the rate of uptake is reduced (Tyler et al. 1990). This phenomena may explain how
uptake of vitellogenin averaged 8. 7% in oocytes < 1 mm while uptake in oocytes
collected from fish near ovulation exhibited a ten-fold reduction in uptake. An
additional factor that may have contributed to the decrease in uptake in latter studies
was the volume of the incubation medium. Oocyte incubations were originally
conducted in 65 p.L of medium in eppindorff tubes. Later incubations were conducted
in culture wells containing 1 mL of medium. Oocytes with diameters of 2 mm would
not have survived 4 hr incubations in only 65 p.L of medium. The proximity of the
vitellogenin ligand to its respective receptor was reduced in latter studies.
39
Aroclor 1260 Residues and Vitellogenic Capabilities in Field Samples
Channel catfish were sampled September through November, 1994, by the
Alabama Department of Environmental Management. Fish were collected through
electrofishing and gill netting of Choccolocca Creek (Logan Martin Reservoir), Cedar
Bluff causeway, and the Alabama-Georgia State line, both on Weiss Reservoir. The
catfish collected from the Coosa River averaged 446 + 20 mm (mean
error) in total length and weighed 957
these fish averaged 0.97
+
± standard
197 g (Table 3). Relative weights (Wr) of
± 0.02; regression analysis revealed a significant increase in
relative weight with increasing length of fish.
Fish from Logan Martin Reservoir were analyzed for PCB residues
individually; however, tissue samples from Weiss Reservoir were composited. Tissue
PCB residues in channel catfish collected from Logan Martin Reservoir were an order
of magnitude greater (34.71
± 13.85 mg/Kg) compared to channel catfish sampled
from Weiss Reservoir (composite sample 1.43 mg/Kg). Serum vitellogenin from
these fish averaged 27.13
± 26.78 mg/mL and 0.020 mg/mL for Logan Martin and
Weiss Reservoirs, respectively.
40
Table 3 Polychlorinated biphenyl (PCB) level in fillets (mg/Kg) and corresponding
serum vitellogenin concentration (mg/mL) in channel catfish collected by the Alabama
Department of Environmental Management. Fish from Weiss Reservoir were
collected at the Alabama/Georgia State line and were composited for PCB
determination.
Length
Weight
VG
(g)
Age
(yr)
PCB
(mm)
(mg!Kg)
(mglmL)
Logan Martin
620
2,850
8
48.56
53.92
Logan Martin
450
960
6
20.86
0.36
Weiss
385
490
Weiss
388
510
Weiss
407
640
-
1.43·
0.02
Weiss
•compos1ted samp,le
400
610
-
Reservoir
In addition to the channel catfish collected by ADEM, fish were sampled
through gill netting Weiss and Logan Martin Reservoirs on the Coosa River and Yates
Reservoir, on the Tallapoosa River, from February through June 1995 (Table 4).
Fish collected from the Coosa River were older (mean age
fish collected on the Tallapoosa River (mean age 3.5
= 4.9 + 0.5 years) than
± 0.3 years).
However, fish
collected from the Coosa River were significantly smaller (mean length 391 + 18 mm;
mean weight 516
+ 77 g) than fish collected from the Tallapoosa River (mean length
471 + 22 mm; mean weight 1,110 + 159 g). Relative weights (Wr) for fish
collected from Weiss and Logan Martin Reservoirs averaged 0.85 + 0.06 while fish
collected from Yates Reservoir expressed relatively good condition (1.01
± 0.06).
Dressout percentage, i.e., the ratio of the fillet weight (g) compared to total body
weight (g), expressed as a percent, was higher for Tallapoosa River channel catfish
compared to channel catfish collected from the Coosa River with 29.31 +0.56 % and
41
23.00
+ 0.98%,
respectively, and reflects the relatively good condition of channel
catfish sampled from the Tallapoosa River.
Polychlorinated biphenyl residues (Table 4) in fish collected through gillnetting Weiss Reservoir averaged 1.51
± 0.59 mg/Kg. Only one fish was collected
from Logan Martin Reservoir, and it contained 7 mg/Kg. Fish collected from Yates
Reservoir contained no detectable PCB residues. Tissue PCB-residues in channel
catfish collected through gill-netting on the Coosa River were significantly correlated
(Pearson Correlation coefficient = 0.47) with the age of the catfish. There was a
positive correlation (Pearson Correlation coefficient = 0.84) between PCB residues
and the relative condition (Wr) of channel catfish sampled from the Coosa River.
There was no correlation between PCB residues and ratios of gonad, kidney, spleen
or liver weights to body weight of the fish (Table 5). There was no correlation
between serum vitellogenin and tissue PCB residues. There was no correlation
between dressout percentage and PCB residues.
The gonadosomatic index was positively correlated (Pearson Correlation
coefficient = 0.63) with serum vitellogenin (mg/mL). There were no detectable PCB
residues in ovaries from channel catfish sampled from Yates Reservoir. PCB residues
in ovaries of channel catfish sampled from Weiss Reservoir averaged 0.06
+ 0.02
mg/Kg; the ovary from the fish collected in Logan Martin reservoir contained 0.23
mg/Kg. In fish from both Weiss and Logan Martin reservoirs, ovaries contained
approximatly 3% of level of PCB residues found in the fillet.
42
Table 4 Aroclor 1260 (A1260) and dichlorodiphenyldichloroethene (DDE)
concentration (ppm) in skinless channel catfish fillets collected from Weiss (Cherokee
County) and Logan Martin (Talladega County) Reservoirs on the Coosa River and
Yates Reservoir (Elmore County) on the Tallapoosa River in Alabama from February
through June 1995. (ns denotes not detected)
Reservoir
Weiss
Weiss
Weiss
Weiss
Weiss
Weiss
Yates
Yates
Yates
Yates
Yates
Yates
Yates
Yates
Yates
Yates
L. Martin
Weiss
Weiss
Weiss
Date
2/18/95
2/23/95
2/24/95
2/27/95
3/03/95
3/05/95
3/10/95
3/10/95
3/10/95
3/10/95
3/10/95
3/11/95
3/11195
3/11/95
4/05/95
4/05/95
4/12/95
4/25/95
6/01195
6/01195
Length
Weight
(mm)
(g)
Age
(yr)
447
343
335
350
420
352
542
477
504
500
405
480
485
365
421
430
440
475
430
315
804
402
247
259
671
310
1885
1253
1746
1582
713
1031
1131
434
701
627
691
754
902
200
6
5
4
5
6
4
4
5
4
4
3
3
2
3
3
4
6
4
7
2
43
A1260
DDE
(ppm)
(ppm)
0.79
4.67
2.00
0.41
1.90
0.83
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
7.00
ns
0.07
0.04
0.10
0.07
0.22
0.88
0.25
ns
0.16
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
.
.
Livers of fish collected from Weiss, Logan Martin and Yates reservoirs were
used to examine in vitro vitellogenic capabilities. Viability of primary hepatocyte
cultures at 24 and 48 hr averaged 59.45
± 7.06% and 54.27 ± 7.53%, respectively.
Vitellogenin in culture media at 24 and 48 hr is presented in Table 6; in YtirQ
synthesis of vitellogenin by hepatocytes was not correlated with PCB tissue residues.
44
Table 5. Age, gonadosomatic (GSI), hepatosomatic (HSI), splenosomatic (SSI),
renalsomatic (KSI) indices, dressout percentage (DOP), and relative weight (Wr) for
channel catfish collected from Weiss, Logan Martin, and Yates Reservoir, February
through June 1995.
Sample
Date
Reservoir
Age
GSI
HSI
KSI
SSI
DOP
Wr
2/18/95
Weiss
6
.019
.033
.007
.002
20.1
1.03
2/23/95
Weiss
5
.079
.021
.005
.008
21.5
1.24
2/24/95
Weiss
4
.009
.032
.009
.001
19.8
0.79
2/27/95
Weiss
5
.014
.025
.008
.001
18.9
0.73
3/03/95
Weiss
6
:021
.035
.006
.001
26.5
1.02
3105195
Weiss
4
.019
.020
.007
.001
23.4
0.84
3/10/95
Yates
4
.093
.023
.006
.001
27.6
1.14
3/10/95
Yates
5
.072
.019
.008
.001
32.8
1.15
3/10/95
Yates
4
.091
.019
.005
.001
28.1
1.34
3/10/95
Yates
4
.006
.014
.005
.001
27.5
0.69
3/10/95
Yates
3
.008
.025
.006
.001
30.7
1.11
3/11/95
Yates
3
.128
.030
.009
.002
27.3
0.93
3/11195
Yates
2
.005
.019
.006
.002
31.2
0.98
3/11195
Yates
3
.024
.024
.007
.001
29.8
0.95
4/05/95
Yates
3
.021
.017
.006
.001
29.0
0.96
4105195
Yates
4
.061
.025
.008
.001
28.7
0.81
4/12/95
Logan
6
.149
.019
.006
.001
25.3
0.82
4/25/95
Weiss
4
.166
.019
.007
.001
27.71
0.70
6/01195
Weiss
7
.075
.019
.008
.001
20.92
0.64
6/01195
Weiss
2
.0001
.013
.008
.001
25.39
0.70
45
Table 6. Serum vitellogenin (mg/mL), primary tissue culture vitellogenin at 24 hr
and 48 hr (mg/mL), Aroclor 1260 tissue residues (ppm) in fillet, and Aroclor 1260
residues (ppm) in ovaries of channel catfish collected from Weiss, Logan Martin, and
Yates Reservoir, February through June 1995.
Sample
Date
Reservoir
Serum
VG
mg/mL
Primary
Culture
24 hr
mg/mL
Primary
Culture
48 hr
mg/mL
A1260
Fillet
(ppm)
A1260
Ovary
(ppm)
2/18/95
Weiss
6.22
.065
*
.079
0.03
2/23/95
Weiss
5.08
*
.075
4.67
0.03
2/24/95
Weiss
0.12
.042
.001
2.00
ns
2/27/95
Weiss
8.08
.033
.037
0.41
0.12
3/03/95
Weiss
*
*
*
1.90
0.06
3/05/95
Weiss
28.29
.022
.049
0.83
ns
3/10/95
Yates
32.80
.271
ns
ns
3/10/95
Yates
*
*
*
.072
ns
ns
3/10/95
Yates
33.27
.024
.043
ns
ns
3/10/95
Yates
.230
.009
.003
ns
ns
3/10/95
Yates
0.24
.009
.003
ns
ns
3/11/95
Yates
*
.006
*
ns
ns
3/11195
Yates
.387
.002
.009
ns
ns
3/11195
Yates
8.58
.002
.005
ns
ns
4/05/95
Yates
36.15
.091
.071
0.00
ns
4/05/95
Yates
36.14
.065
*
0.00
ns
4/12/95
Logan
41.80
.065
.116
7.00
0.23
4/25/95
Weiss
21.90
.145
.122
0.00
ns
6/01195
Weiss
28.65
.015
.035
0.07
ns
6/01195
Weiss
samp>le m1ssmg
ns not detected
0.60
.015
.035
0.04
ns
46
Isolation and Characterization of Channel Catf"Ish Oocyte Vitellogenin Receptor
Vitellogenin receptor from channel catfish oocytes was isolated and characterized.
The putative receptor, isolated through extraction with octyl-{j-D-glucoside and visualized
with ligand blotting, has a molecular mass of 100 kDa. Ligand blotting using
125
1-labeled
vitellogenin confirmed that the single 100 kDa band was binding vitellogenin. Ligand
blotting with increasing levels of vitellogenin exhibited a positive dose response (Figure 10).
Vitellogenin binding was blocked by sodium suramin at concentrations greater than 0.1mM.
lnterspecies Comparisons of 17{j-estradiol-induced Serum Proteins Reactivity to Monoand Polyclonal Antibody
Work has also been conducted which examined the reactivity of 17{j-estradiol-induced
proteins in serum from several species of fish to both polyclonal and monoclonal antibodies
reactive against channel catfish vitellogenin. Both monoclonal and polyclonal antibodies
were reactive in Western blot against serum from channel catfish, blue catfish, flathead
catfish, and brown bullhead. Figure 10 shows an SDS PAGE gel containing serum from
spottail gar, common carp, flathead catfish, smallmouth buffalo and white catfish; unlike
serum from other fish, the white catfish had not been induced with 17{j-estradiol. Only lanes
containing serum from flathead and channel catfish were reactive on Western blot using
monoclonal antibody. A similar Western blot using polyclonal antibody (Figure 12) shows
that neither spottail gar nor common carp reacted; however, flathead catfish, smallmouth
bufallo, and white catfish were reactive using polyclonal antibody. While protein bands in
the 150 kDa range were reactive, the most intense reaction was associated with protein
aligned with the 66 kDa molecular mass marker. Neither the polyclonal nor the monoclonal
47
antibodies reacted against 17{3-estradiol-induced serum from redear sunfish nor white bass.
Additionally, flathead catfish 17{3-estradiol induced serum protein was shown to bind the
channel catfish vitellogenin receptor (Figure 13). The reactivity of the monoclonal antibody
to vitellogenin of other catfish species broadens the number of species over which the effects
of environmental pollutants on hepatic synthesis of vitellogenin can be examined with this
antibody.
48
Figure 10. Coomassie blue R stained gel containing serum collected from fish before and
after induction with 17{j-estradiol. Lanes 3 and 4 contain serum from pre and post-17{jestradiol-induced serum from spottail gar, respectively. Lanes 5 and 7 and lanes 6 and 8
contain pre and post-induced serum from common carp, respectively. Lane 9 contains
preinduced flathead catfish serum; lanes 10 and 11 contain postinduced flathead catfish
serum. Lane 12 contains induced smallmouth buffalo serum, and lane 13 contains white
catfish serum. Lanes 1 and 20 contain high molecular mass markers, and lanes 2 and 14
contain channel catfish vitellogenin standards.
49
14 13 li II I 0 9 8
-
1 i
5 4 3
z.. . . .
~
.....
t
Figure 11. Western blot, using monoclonal antibodies, of gel containing serum from fish
before and after induction with
17~-estradiol.
Lanes 3 and 4 contained serum from spotted
gar; lanes 5 through 8 contained serum form common carp; lanes 9 through 11 contain serum
from flathead catfish, lane 12 contained serum from smallmouth buffalo, and lane 13
contained serum from white catfish (uninduced). Lanes 2 and 14 contained channel catfish
vitellogenin standards.
so
Figure 12. Western blot, using polyclonal antibodies, of gel containing serum from fish
before and after induction with 17(j-estradiol. Lanes 3 and 4 contained serum from spotted
gar; lanes 5 through 8 contained serum form common carp; lanes 9 through 11 contain serum
from flathead catfish, lane 12 contained serum from smallmouth buffalo, and lane 13
contained serum from white catfish (uninduced). Lanes 2 and 14 contained channel catfish
vitellogenin standards.
51
7
205
VG)IIIIoo..
116
97
9
I0
II
I~
13
.•
..
.. .
lil~l~l:llll
- .. .. ... - - . .
......
~. ·~
... - . "
14 15,
~P'IIIIII
. .~ .
~:;~
...
43
9
~
;.;
.
J
.
Figure 13. Coomassie blueR stained gel containing serum from fish before and after
induction with 17{:1-estradiol. Lanes 3 and 13 and lanes 4 and 12 contain serum from pre and
post-17{:1-estradiol-induced serum from white bass, respectively. Lanes 5, 7, 9 and 11 and
lanes 6, 8, and 10 contain pre and post-induced serum from redear sunfish, respectively.
Lanes 1 and 15 contain high molecular mass markers, and lanes 2 and 14 contain channel
catfish vitellogenin standards (VG).
52
~--:,
',.--.,-
10 1tJ
....--:
,
..
50 50
r)
~
I
.
.-~
--:
-~l
~ 1100
I
:
~~.,
. .I
..,..
!
I
1
I
\~:
I -
I
lI
---------
~
.
Figure 14. Ligand blot of octyl-~-D-glucopyranoside extract of channel catfish oocyte
membrane preparation incubated with 0, 1, 50, and 100 p.L of sera from
induced flathead catfish.
53
17~-estradio1
Discussion
The effects of many chemical contaminates and their ability to bioaccumulate are
dependent on their lipid solubility and on the extent to which the compound is subject to
biotransformation. Through the process of biotransformation, the major site of which is the
liver, a variety of lipophillic compounds are rendered more water soluable to facilitate their
excretion. During this process compounds undergo structural changes that may result in
reactive intermediates. The reactive intermediates, eg. free radicals, may in tum interfere
with normal biochemical pathways to alter their outcome.
Lipids represent a source of fuel for the liver's biotransforming and synthetic
pathways and they function as part of the post-translational modification performed by the
liver. One such biosynthetic pathway that utilizes lipids as both fuel and as a structural
component is estrogen-induced vitellogenesis. Vitellogenin-derived yolk protien is the
primary source of nutrients and energy for the embryonic development of most fish. This
study examined the effect of Aroclor 1254, a lipophilic compound representing a group of
polychlorinated biphenyls, on the hepatic synthesis of the lipoprotein, vitellogenin, and on the
receptor-mediated uptake of vitellogenin by oocytes. Here we report decreased levels of
vitellogenin in primary cultures of channel catfish hepatocytes treated with Aroclor 1254.
The ultimate effect of decreased hepatic synthesis of vitellogenin may be a reduction in
reproductive success resulting from fewer eggs or diminished yolk for developing embryos.
Because the liver is the major site of biotransforming enzyme systems, isolated
hepatocytes have been used to study the metabolism of various xenobiotics. Metabolism of
xenobiotics by isolated fish hepatocytes in primary culture closely approximates in vivo
54
metabolism patterns (Nishimoto et al. 1992). Both Phase I and Phase II reactions have been
demonstrated in fish with various enzyme activities qualitatively similar to those found in
mammals (Vodicnik et al. 1981; James and Bend 1980). However, the use of fish
hepatocytes in primary culture has presented difficulties as cells dedifferentiate over time and
lose cell-specific functions (Flouriot et al. 1993). This study has documented in vitro
synthesis of vitellogenin by channel catfish hepatocytes in primary culture, induced in vivo
with 17{3-estradiol, through incorporation of 33P-label. The 150 kDa-protein exhibited
electrophoretic mobility and immunological cross-reactivity identical to that described for
putative vitellogenin produced by liver explants from estradiol-treated male channel catfish
(Bradley and Grizzle 1989).
Aroclor-treated hepatocytes contained significantly less vitellogenin than acetone
treated hepatocytes or hepatocytes receiving no chemical treatment. Several mechanisms
have been suggested to account for decreased levels of vitellogenin from down regulation of
the estrogen receptor (Romkes et al. 1987; Astroff and Safe 1988) to increased metabolism
of 17{3-estradiol through induction of hepatic cytochromes P-450 (Spink et al. 1990). While
it was not within the scope of our study to determine the mechanism through which Aroclor
exerted its effect on vitellogenin levels, it is interesting to note that induction of the
cytochrome P450 enzyme systems that has been demonstrated with Aroclor pretreatment
(Smith et al. 1990; Thomas 1988; Forlin and Haux 1985) has also been associated with the
formation of reactive intermediates that are capable of disrupting a number of cellular
activities. Additionally it is reasonable to believe that the induction of the P450 enzymes
may reduce the cellular machinary available for vitellogenesis just as the synthesis of serum
55
protein is inversely related to induction of vitellogenesis (Kwon et a1.1993; Stanchfield and
Yager 1978). Regardless of the mechanism, Aroclor treatment has been demonstrated to
affect vitellogenin levels in vitro and in vivo (Chen et al. 1985). Studies have demonstrated
an inverse relationship between the total concentration of PCBs in eggs and hatching success
of the fish (Ankley et al. 1991) and that high body burdens of lipid-soluable organochlorines
such as PCBs were associated with reduced reproductive capability in fish (Monosson et al.
1994; Norrgren et al. 1993; Mac and Edsall 1991; Andersson et al. 1988; Mac 1988; Hansen
et al. 1985; Westemhagen et al. 1981). The effect of these chemical contaminants in fish
has in tum prompted a legitimate concern over the possible health threat to people consuming
these fish (Fein et al. 1984; Cordle et al. 1982).
In the second year of the study the effects of Aroclor 1254 on receptor-mediated
uptake of vitellegenin by channel catfish hepatocytes was examined. Aroc1or 1254 did not
appear to effect receptor-mediated uptake of vitellogenin by oocytes in YitrQ. However,
serum lipoproteins similar to vitellogenin have been suggested to be carrier molecules for
hydrophobic compounds and may represent a mechanism through which these compounds
translocate to the gonads and affect reproduction (Placket al. 1979). Studies have revealed
that ovaries can contain as much as 2.5 times greater the PCB concentration of dorsal muscle
(Ankley et al. 1991) and that relative to other congeners, the more toxic congeners of PCBs
are enriched in the oocytes (Miller 1993; Ankley et al. 1989).
Incubations of oocytes with increasing concentrations of Aroclor 1254 exhibited a
trend of increasing uptake which only proved significant in 1 of the 3 studies. While two of
the studies failed to show significant differences in uptake between treatment groups, the
56
third study revealed that oocytes treated with 111 and 260 p.g Aroclor had significantly
higher uptake than oocytes treated with 9 p.g Aroclor. These results were confounded by
studies showing that oocytes treated with suramin and increasing concentrations of Aroclor
also exhibited an increase in uptake. Data from suramin treated oocytes suggest that the
observed increase in 'uptake' with increasing concentrations of Aroclor is a result of some
mechanism other than receptor-mediated uptake. The difficulty in working with oocytes
divested of their outer follicular layers was problematic throughout the second year of the
study. Recent work has revealed however, that ovarian follicles cultured within lamellae had
a two-fold increase in survival compared to oocytes divested of their outer follicular layers
(Nagler et al. 1994). While oocytes divested of their outer epithelium had increased rates of
vitellogenin uptake, the procedure decreased survivability of the oocytes.
In the third year of the study PCB residues in channel catfish collected from Logan
Martin and Weiss Reservoirs were within the range, i.e., 0 to 260 p.g/mL, examined in Year
1 and 2 of the study; however, in vitro culture of hepatocytes from fish collected in Year 3
did not exhibit reduced vitellogenin capabilities relative to tissue PCB residues. In vivo
studies of white perch (Morone americana) injected with one congener of PCB, 3,3' ,4,4'tetrachlorobiphenyl, also failed resolve any effect on serum vitellogenin levels (Monosson et
al. 1994). This discrepancy may be related to the relative potentcy of the congeners under
study. It should be noted that the in vitro studies conducted in Year 1 and 2 of this study
focused on the effect of Aroclor 1254. The congener pattern in wild fish was similar to that
of Aroclor 1260 that was used as a reference standard.
57
PCB residues in wild fish were significantly correlated with the age and size of the
fish. Similar results were obtained through regression analysis of PCB residues in fish
collected by ADEM since 1978 on Weiss Reservoir where a significant amount of the
variability in the data was accounted for through the use of age and year of collection (data
not shown). Studies on the toxic effects of pesticides have revealed that organisms often
develop a resistance and that pesticides in combination with other chemical contaminates may
exhibit effects contrary to those produced in the laboratory.
Although vitellogenesis is a potential model for examining the effects of
environmental xenobiotics on fish reproduction (Chen et al. 1985; Chen 1988; Spies et al.
1990); in vivo assays involve considerable time. The ability to detect inhibition of
vitellogenin synthesis by hepatocytes exposed to Aroclor 1254 in vitro provides an assay with
which to screen other environmental pollutants. This technique affords a means of rapidly
predicting the extent to which chemical residues affect hepatic function and reproduction in
native fishes.
In addition, this study has demonstrated that both monoclonal and polyclonal
antibodies were reactive against 17(3-estradiol-induced serum protein in channel catfish, blue
catfish, white catfish, flathead catfish, yellow bullhead, and brown bullhead. Neither the
polyclonal nor the monoclonal antibodies reacted against 17(3-estradiol-induced serum protein
from spotted gar, common carp, redear sunfish, nor white bass. The polyclonal antibody did
react with serum proteins from both catfish and smallmouth bufallo. These data indicate that
the vitellogenin gene is highly conserved within family Ictaluridae; however, fish families
closely related to the catfishes, eg. Catastomidae and Cyprinidae, did not react to the
58
monoclonal antibody. Additionally, the various fish vitellogenins were used for ligand
blotting with the isolated channel catfish oocyte membrane vitellogenin receptor. Flathead
catfish serum was shown to bind the channel catfish vitellogenin receptor. The reactivity of
other catfish vitellogenins to the monoclonal antibody broadens the potential for examining
the effects to environmental pollutants on hepatic synthesis of vitellogenin.
In conclusion, the project objective of Year 1 of the study was successfully
accomplished; primary cultures of hepatocytes treated with Aroclor 1254 at concentrations of
9 to 260 mg/L contained less vitellogenin than their untreated counterparts. Studies of
hepatic vitellogenesis demonstrated the usefulness of this in vitro technique to assay effects of
Aroclor 1254 on vitellogenesis. In Year 2, radiolabeled vitellogenin was isolated and used to
demonstrate uptake of vitellogenin in vitro. Results from suramin-treated oocytes suggested
that the observed increase in uptake with increasing concentrations of Aroclor was a result of
some other mechanism than receptor-mediated uptake. Polychlorinated biphenyl residues in
fish collected in Alabama were consistent with levels examined through in vitro studies
conducted in Years 1 and 2; however, there was no correlation between vitellogenin serum
vitellogenin or vitellogenin in primary culture media and PCB residues. The failure of work
conducted in Year 3 to collaborate work conducted in vitro keynotes the value of in vitro
studies. Aroclor 1254 has been associated with diminished levels of vitellogenin both in
vitro and in vivo. The inability to discern this effect in samples collected from the wild
suggests that the effect is obscured compared to when studied directly in a laboratory
environment.
59
The ability to detect hepatic vitellogenesis in vitro and its sensitivity to Aroclor 1254
provide an assay with which to screen other environmental pollutants. This technique affords
a means of predicting the extent to which chemical residues affect hepatic function and
reproduction in native fishes.
Acknowledgments
Preliminary work on this study was accomplished through funds provided by the U.
S. Fish and Wildlife Service. Special thanks are given to the Water Resources Institute,
Auburn University, Alabama, for providing support for the continuation of this work. The
authors would like to thank Dr. J. L Sartin, Jr., and Ms. Barbara P. Steele for providing
[
125
1]-iodide and for conducting the labeling procedure. We thank Ms. Barbara Estridge for
her assistance with the initial experiments examining
125
I-vitellogenin uptake by channel
catfish oocytes. We thank Mr. John C. Dennis for helping define much of the protocol used
in the isolation and characterization of the vitellogenin receptor. Finally we wish to thank
Mr. Oscar D. LeCompte, Director of the Alabama Department of Agriculture and Industries
Pesiticide Residue Laboratory Division at Auburn University, and his staff, for their analysis
of PCB residues in the flesh and ovaries of field collected fish.
60
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