Download GnRH Protein Levels in Atrazine-Treated Axolotls

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Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences
2008
GnRH Protein Levels in Atrazine-Treated Axolotls
(Ambystoma mexicanum)
Sara Nienaber and Sarah Leupen
Department of Zoology, Ohio Wesleyan University, Delaware, OH, 43015
Atrazine is the most widely used agricultural herbicide in the United States and a known
endocrine disruptor. In amphibians, it has been shown to cause gonadal malformations,
feminization of males, behavioral changes, and immune suppression; however, its mechanism of
action is unknown. We hypothesized that atrazine reduces the production of gonadotropin
releasing hormone (GnRH) in the hypothalamus. Axolotls (Ambystoma mexicanum) were
exposed to atrazine, 10-8 M ß-estradiol-3-benzoate, or no treatment and were sacrificed at 6, 8,
and 10 months of age. GnRH neurons were labeled using immunocytochemistry, and labeled
neurons were then counted using confocal microscopy. Although no significant difference was
found in the total number of GnRH neurons, ectopic GnRH expression was seen in some brains.
A significant negative correlation was found between presence of ectopic GnRH and number of
normal GnRH neurons. Atrazine-treated animals were more likely than control or estrogentreated animals to have ectopic GnRH expression. The data implicate a central site of action of
atrazine.
Keywords: amphibians, herbicides, endocrine disruption, hypothalamic-pituitary-gonadal axis,
aromatase, estradiol
Introduction
Atrazine is the most widely used
agricultural herbicide in the world, with more
than 60 million lbs used annually in the United
States alone (Hayes et al., 2002). Atrazine was
long considered safe because it is not as directly
toxic as many other agricultural chemicals.
More recently, however, atrazine has been
characterized as an endocrine disrupting
compound (EDC): a chemical that mimics the
action of a hormone, often at very low doses
(Hayes et al., 2002).
Many studies have focused on the
effects of atrazine on amphibians. Due to their
partially aquatic lifestyle and environmental
sensitivity, these animals are especially
vulnerable to contaminants in their habitat (Rohr
and Palmer, 2005). Many amphibian breeding
seasons occur in the spring, when agricultural
chemical application is at its peak, making
young, developing amphibians even more
susceptible to the effects of endocrine disruptors
(Freeman et al., 2005).
The Environmental Protection Agency
sets the maximum amount of atrazine allowed in
drinking water at 3 ppb (Brodkin et al., 2007).
However, malformations and abnormalities in
amphibians have been seen at as low as 0.10 ppb
(Hayes et al., 2002).
Abnormalities in amphibians following
exposure to atrazine include hermaphroditism
and other gonadal abnormalities (Coady et al.,
2005; Hayes et al., 2002; 2006), changes in
testosterone levels (Hecker et al., 2005), poorly
developed larynxes in male frogs (Coady et al.,
2005; Hayes et al., 2006), hyperactivity and
other behavioral changes (Rohr and Palmer,
2005), and immune system suppression
(Brodkin et al., 2007; Houck and Sessions,
2006). Some of these malformations appeared a
year or more after exposure had ceased (Rohr et
al., 2006).
Because of atrazine’s effects, it, along
with other EDCs, has been named a factor in the
global decline of amphibian populations (Rohr
and Palmer, 2005). Atrazine’s mechanism of
endocrine disruption is unknown. Because it
raises levels of circulating estrogens in all
animals studied so far, it has been hypothesized
that activation of the enzyme aromatase, which
converts testosterone into estrogen, may be its
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GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum)
2008
mechanism of action (Hayes et al., 2006).
However, several studies have shown no effect
of atrazine on aromatase (Hecker et al., 2005;
Murphy et al., 2006).
Though possible mechanisms of
endocrine disruption, such as aromatase
activation, have been previously investigated,
few studies have focused on the central effects
of atrazine and other endocrine-disrupting
compounds. Gonadotropin Releasing Hormone
(GnRH) is the master hormone of the
reproductive axis in all vertebrate animals. Its
pattern and level of release from the
hypothalamus dictate the activity of the entire
system. As neuroendocrine controllers, these
GnRH neurons receive not only direct feedback
from gonadal steroids as well as pituitary
gonadotropins, but also an array of sensoryderived neural information communicating the
season (Doer-Schott and Dubois, 1978; Ralph,
1978), the metabolic state (Schneider, 2004;
Wade and Schneider, 1992) and immune status
of the animal (e.g. Bouret et al., 2004; Igaz et
al., 2006), physiological stress (e.g. Moore and
Zoeller, 1985), presence of potential mates
(Boehm et al., 2005; Propper and Moore, 1991),
and other factors.
We hypothesized that atrazine exposure
may reduce the production of GnRH in the
hypothalamus of developing amphibians,
resulting in disrupted steroidogenesis and sexual
development. We chose the axolotl, Ambystoma
mexicanum, as our model organism because its
long history as an experimental amphibian
provides many well-established laboratory
protocols, and because it is large, hardy, and
aquatic throughout its lifecycle.
Materials and Methods
Animal Care
The following protocols were
approved by Ohio Wesleyan University's
Institutional Animal Care and Use Committee.
At four months of age, axolotls (Ambystoma
mexicanum) were divided into three groups of
20 animals each: control, atrazine-treated, and
estradiol-treated. These animals are neotenic
salamanders, which means that they remain
aquatic through their entire lifecycle, and never
metamorphose into terrestrial adults. Four
months of age was chosen as the starting point
for this study because we wanted to begin
atrazine exposure during development, while
GnRH-producing neurons were being formed.
The control group was raised in 40%
Holtfreter’s solution, a slightly saline solution
that is the standard bowl water for axolotl care.
A second group of animals were raised in 3 ppb
atrazine (Supelco, Bellefonte, PA) in 40%
Holtfreter’s solution, which corresponds to the
maximum amount of atrazine allowed in
drinking water by the EPA. Because atrazine
has been shown to elevate estrogen levels,
possibly by inducing aromatase, a third group
was raised in 10-8 M ß-estradiol-3-benzoate
(Sigma Aldrich, St. Louis, MO) in 40%
Holtfreter’s solution. If atrazine’s effects are
mediated in full by increased estrogen levels,
similar effects should be seen in atrazine- and
estradiol-treated animals. By contrast, if atrazine
has effects independent of estrogen activation,
these effects ought to be revealed by direct
comparison of atrazine- and estradiol-treated
groups.
Animals were housed in 2-L plastic
containers and fed salmon pellets ad libitum.
Treatment water was changed as needed.
Axolotls were initially housed three to a bowl,
then slowly transitioned to living in pairs, and
then alone. This shift was necessary as they
grew and in some cases in response to
cannibalistic behavior, which occurs frequently
in juvenile animals of this species. By eight
months of age, all animals were living alone in
2-L containers.
Arbitrarily chosen subsets of animals,
representing small, medium, and large
specimens from each group, were sacrificed by
decapitation at 6, 8, and 10 months of age,
respectively. Previous, unpublished observations
from our lab indicate that GnRH neurons are
formed in most juvenile axolotls between 6 and
10 months of age. Thus, this range of ages was
chosen as the ideal range to reveal potential
differences in GnRH development among the
groups. The heads were preserved in 4oC 4%
paraformaldehyde, as were the weighed gonads
of animals 8 and 10 months of age; gonads of
animals sacrificed at six months of age were not
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Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences
2008
sufficiently differentiated from associated renal
tissue to be collected.
fraction of body weight) could be calculated for
each animal.
Brain Preparation
After at least 24 hours of fixation in
paraformaldehyde at 4oC, brains were removed
from the heads and set in small blocks of 5%
agarose (Fisher Chemical, Fair Lawn, NJ).
Brains were sliced with a vibrating microtome
(Vibratome Co., St. Louis, MO) into 80 µm
slices. The slices were then collected into sixwell plates in phosphate buffered saline (PBS),
pH 7.2.
Immunocytochemistry was then
performed as follows to identify the GnRH
neurons. Slices were blocked in 1% goat serum
in PBS for 30 minutes, and GP6 primary
antibody (generously donated by Lothar Jennes)
was added at a concentration of 1:500 and
incubated for 72 hours at 4oC. Samples were
then rinsed three times with PBS plus 0.3%
triton (PBS-T), and Biotin SP-conjugated goat
anti-guinea pig secondary antibody (Jackson
ImmunoResearch, West Grove, PA) was added
for two hours at a 1:500 concentration. After
three further rinses in PBS-T, samples were
incubated in fluorescein DTAF Streptavidin
(Jackson ImmunoResearch) at 1:500 for one
hour.
After rinsing samples three times with
PBS-T, brain slices were mounted onto slides
and confocal microscopy was used to count the
number of GnRH neurons in each brain. Slides
were coded with neutral names, so that the
person counting the neurons was blind to
experimental group until the completion of the
study. The number of normal GnRH neurons in
each section was counted, and any abnormalities
in GnRH expression were also recorded.
Minitab and Excel were used for calculations of
t-tests and analysis of variance (ANOVA) when
appropriate.
Results
The GnRH signal was more complex
than expected. In addition to normal GnRH
neurons (Fig 1, top), a subset of brains displayed
additional specific fluorescent labeling
(“ectopic” GnRH expression; (Fig 1, bottom).
Normal GnRH neuron
Ectopic GnRH
Figure 1: Digital images of GnRH fluorescence
in axolotl brains. Top, a normal GnRHproducing neuron; bottom, ectopic GnRH
expression.
Other measurements
Monthly weights were recorded for each
animal. At the eight and 10 month sacrifice
dates, gonads were also removed from and
weighed for each animal sacrificed so that the
gonadosomatic index (GSI; gonad weight as a
Figure 2: Proportion of animals per group with
ectopic GnRH expression. Ectopic GnRH was seen
in animals of all treatment groups, but with greatest
frequency in the group of axolotls treated with
atrazine (p=.005 vs. control, p=0.03 vs. estradiol).
No previous literature mentioned the
presence of ectopic GnRH which was surprising
considering the frequency of this occurrence
among our experimental animals. Though it
may have been useful to somehow quantify the
areas of ectopic GnRH, there was no satisfying
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GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum)
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Differences in proportion of animals
displaying ectopic GnRH were calculated
using a z-test for proportions. As indicated
in Figure 2, ectopic GnRH expression was
predominantly seen in atrazine-treated
animals (11 of 14), significantly more so
than in control (2 of 12, p=0.003) or
estradiol-treated (3 of 9, p=0.04) animals.
There was no significant difference between
estradiol-treated and control animals.
100
25
Normal GnRH Neurons
(mean number/individual)
or objective way to do so, because ectopic
GnRH usually occurred in branching streaks or
overlapping blocks, rather than being separated
into distinct elements.
20
15
10
5
0
Control
Estradiol
Atrazine
Figure 3: Mean number of normal GnRH neurons
per axolotl brain for control, estradiol-treated,
and atrazine-treated groups. There were no
significant differences among the groups
(p=0.10).
80
70
60
50
40
30
20
10
0
Control
Estradiol
Atrazine
Figure 2: Proportion of animals per group with
ectopic GnRH expression. Ectopic GnRH was seen
in animals of all treatment groups, but with greatest
frequency in the group of axolotls treated with
atrazine (p=.005 vs. control, p=0.03 vs. estradiol).
A one-way ANOVA was performed to
compare the number of normal GnRH neurons
across treatment groups. There was no
significant difference in mean number of
normal GnRH neurons per axolotl among
treatment group(s) (Figure 3, Fdfb,dfw=32,
p=0.10); although a trend is apparent, it is not
statistically significant, due to the large variation
in number of neurons observed within groups.
A one-way ANOVA was performed to
compare the number of normal GnRH neurons
in brains with and without ectopic GnRH. As is
indicated in Figure 4, there were significantly
fewer GnRH neurons in those brains with
ectopic GnRH than those without, regardless of
treatment group (Fdfb,dfw=33, p=0.02).
20
18
16
Normal GnRH Neurons
(total number)
% Animals with Ectopic GnRH
90
14
12
10
8
6
4
2
0
Brains with Ectopic GnRH
Brains without Ectopic GnRH
Figure 4: Brains expressing ectopic GnRH had
fewer normal GnRH neurons (p=0.02) than
those without ectopic GnRH.
Repeating this ANOVA analysis using
only atrazine-treated animals, the same pattern is
revealed, though the groups are not significantly
different (Fdfb,dfw=12, p=0.10).
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Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences
2008
Normal GnRH Neurons (total number)
20
18
16
14
12
10
8
6
4
2
0
Brains with
and without ectopic GnRH
Figure 5: Brains expressing ectopic GnRH had
fewer, though not significantly (p=0.10),
normal GnRH neurons than those without
ectopic GnRH.
Due to the great variation in size of
axolotls of the same age which normally occurs,
no difference was seen in the weight of animals
in each treatment group over the course of this
study (in all monthly comparisons p=NS).
Because gonadal abnormalities have been seen
regularly in male amphibians exposed to
atrazine (ex. Hayes et al., 2002), gonadosomatic
index was calculated for each animal sacrificed
at eight and ten months of age. Prior to this age,
most gonadal tissue was too poorly
differentiated from renal tissue to be measured
accurately. No differences were seen among the
gonadosomatic indices of the treatment groups.
Full characterization of gonadal morphology
will require histological work with the isolated
gonadal tissue.
Discussion
An underappreciated danger of EDCs is
that they defy the basic principles of toxicology;
larger doses do not necessarily produce greater
harm (Hayes et al., 2002). Rather, minute
amounts of a compound can have dire
consequences for an organism. Many of these
compounds, like atrazine, produce few if any
outward malformations, so a chemical may be
on the market for decades before it is connected
to a problem such as global amphibian
population declines. With so many chemicals
on the market and many more being introduced
annually, it is impossible to monitor the safety of
each compound, especially if compounds are
assumed to be safe until proven otherwise.
It is important to understand the
mechanisms by which chemicals such as
atrazine act as endocrine disruptors, so that
newly developed chemicals may be designed to
minimize endocrine disruption. GnRH is an
important target to examine as a possible site of
endocrine disruption, since endocrine disruptors
(including atrazine) have been shown to cause
sexual malformations, immune disruption, and
behavioral changes, all of which could
conceivably be downstream of changes in GnRH
release, and more directly, because gross
abnormalities in reproductive function are likely
to be attributable to a disruption “high” within
the reproductive axis.
The most surprising result of this study
was the occurrence of ectopic GnRH, which was
previously undocumented. Our results indicate
that the presence of ectopic GnRH occurs rarely
in axolotls under normal conditions. However,
exposure to chemicals such as atrazine may
increase the likelihood of this occurring. The
presence of ectopic GnRH was associated with a
reduced number of normal GnRH neurons,
which may lead to changes in gonadotropin
synthesis and release, endogenous sex steroid
production, gametogenesis, and sexual behavior.
Since the presence of ectopic GnRH was a
surprise finding in this study and has not
previously been documented, it may be
beneficial to design further studies to investigate
what this ectopic GnRH actually is, how it
forms, and how to quantify its production. A
challenge of this study was that the amount of
ectopic GnRH could not be easily measured,
because it occurs in streaks rather than welldefined cell bodies.
In the future, other methods of
measuring GnRH levels could be used to
quantify GnRH expression or release. One such
method is an ex vivo assay, which has previously
been used to measure GnRH release in
amphibians (Tsai, Moenter, and Cavolina,
2003). This protocol involves hypothalamic
perfusion and frequent sampling in order to
assay the GnRH release of the tissue. An
advantage of this method is that it measures
GnRH release directly, rather than protein
levels, which may or may not be directly
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GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum)
2008
correlated with release. However, this method
does not allow for observations of the
anatomical pattern of GnRH expression, the
presence of ectopic GnRH, or the existence of
other abnormalities at the cellular level.
Though this experiment began with 20
individuals in each group, cannibalism of bowlmates reduced the number of individuals in each
group. Despite the fact that it may be more
labor intensive, in the future it may be useful to
house animals individually from the beginning,
to prevent cannibalism.
It is possible that atrazine affects the
GnRH expression of males differently than
females. Indeed, sexual malformations in
response to atrazine have thus far only been
found in male amphibians. We would ideally
like to examine our data separately by sex.
However, many of our animals were too young
or too early in sexual development to accurately
determine sex, and genetic or chromosomal sex
determination methods for axolotls do not exist.
In future studies, it may be beneficial to raise all
specimens into adulthood, and divide the
animals up by sex to process the data. Of
course, further perturbations of the
hypothalamic-pituitary-gonadal axis may also
occur in older animals, or conversely, earlier
perturbations may diminish.
This study did not establish atrazine’s
mechanism of action as an EDC, but the data are
consistent with an aromatase-inducing or other
estrogen-stimulating action. Estradiol treatment
was expected to inhibit GnRH expression and
therefore lower the number of countable GnRH
neurons, through physiological negative
feedback in the hypothalamo-pituitary-gonadal
axis. Although it did not statistically
significantly do so, the similar number of mean
GnRH neurons in the estradiol- and atrazinetreated groups is consistent with this
interpretation. Importantly, independent of the
“aromatase question”, which predicts that
atrazine exposure up regulates aromatase, and
therefore conversion of testosterone to estradiol,
this is the first study to demonstrate that atrazine
may effect the morphology of the central
nervous system.
The significance of ectopic GnRH
expression is unclear. The morphology suggests
induced GnRH production by endothelial cells
in cerebral blood vessels. This may represent a
generalized stress or immune response, which
could be induced or exacerbated by exposure to
a stressor such as a chemical pollutant.
However, it is also possible that the induced
peptide is not GnRH, but rather another peptide
which is cross-reacting with the GnRH antibody.
No previous studies have reported cross-reacting
of the antibodies used in this experiment, so it is
assumed that the likelihood of this occurring is
low.
It is important to delineate the
developmental window of harm by exposure to
atrazine in developing amphibians. What dose,
for what duration, at what time in development,
maximizes (or minimizes) central and peripheral
reproductive disruption, and by what
mechanism? Future work will prioritize these
questions.
Acknowledgements
We would like to thank the Support of
Mentors and their Students in the Neurosciences
(SOMAS) for their funding of this work, as well
as the Ohio Wesleyan University Summer
Science Research Program (SSRP) for their
support of this project. We are very grateful to
Lothar Jennes for the generous donation of the
GP6 primary antibody, and to Katie Ayers and
Tov Nordbo for their assistance in animal care
over the course of this project.
Corresponding Author
Sara Ann-Marie Nienaber
Ohio Wesleyan University
Email: [email protected]
Sara Nienaber
HWCC Box 1931
Delaware, OH 43015
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2008
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