The Effect of Early Androgen Exposure on Adult Zebrafish
Behavior
Introduction
Testosterone is the primary male sex hormone found in almost all vertebrates. It
is a steroid hormone derived from cholesterol and has been found to be responsible for
several functions and side effects - one of these side effects is aggression. Several
studies (Tollman & King, 1956), (Persky et al., 1971), (Van de Poll et al., 1988) have
found evidence correlating testosterone and aggression. Numerous studies find the
hormone exerts its effects on the individual during the first few days after birth. It is
during this time the neural network grows and forms connections the most rapid and is
therefore vulnerable to surrounding molecules such as hormones (Sluyter et al., 1994).
Studies have recently focused on how early exposure to exo- and endogenous
substances influences adult phenotypes. In zebrafish, early developmental factors such
as alcohol (Fernandes & Gerlai, 2009), metals (Powers et al., 2010), and abnormal
lighting (Bilotta, 2000) have been shown to affect adult behavior.
The conversion of testosterone to 5-alpha-dihydrosterone (5- -DHT) is believed
to be the key biochemical process that leads to aggressive behavior in males. In
females, it is the aromatization of testosterone to estradiol. These conversions lead to
the newly formed hormones, 5- -DHT and estradiol, to act respectively on the androgen
or estrogen receptors in the brain. Evidence linking aggressive behavior with high
aromatization and estrogen receptor levels is what led to these conclusions (Schlinger &
Callard, 1990).
Estradiol is the principal female sex hormone. It, like testosterone, is found in
both sexes and is a steroid hormone derived from cholesterol. A portion of estradiol in
the body is gained from the aromatization of testosterone. Studies have also
discovered a correlation between estradiol levels and aggression (Trainor et al., 2008).
Due to these studies, along with the findings that elevated estrogen receptors in the
brain correlate with high aggression levels, estradiol has become a primary focus in
aggression studies.
Male fish have a different foremost sex hormone. 11-ketotestosterone serves in
their spermatogenesis (Nagahama et al., 1994). However, testosterone and estadiol is
present in both females and males. In this study, I examine the effect of early exposure
of these hormones on adult zebrafish behavior. I exposed zebrafish eggs to
testosterone and estrogen and measured both their aggression and avoidance levels.
Materials and Methods
Zebrafish (Danio rerio) were chosen as a focal species for a number of reasons.
They are an important model organism for research and can correlate well to human
health issues, large numbers can easily be housed, and they are sessile for their first
five days of development, which makes observations easy. Adult zebrafish (Danio
rerio) were obtained from Aquatica Tropicals Inc. (Plant City, FL) and populations of
wildtype and long fin gold were maintained according to standard procedures
(Westerfield 1994). One hundred sixty-two eggs were collected from a mass breeding
and randomly divided among nine different 250 ml Erlenmeyer flasks: three flasks
containing a 1.0 M solution of testosterone, three containing a 1.0 M solution of
estradiol, and three containing a normoxic water solution which would act as the control.
The flasks were incubated in a temperature controlled bath at 27 ˚C for 24 hours.
At the end of the 24 hours, the eggs were transferred to separate 2 L containers
supplied with a common water source at 27 ˚C for the remainder of development (ZMod housing system, Marine Biotech, Beverly, MA). . Newly hatched larvae were fed 23 times daily and kept in these conditions for 150 days when the assay was performed.
Behavioral assay
Individual fish were placed into a 30 x 30 x 30 cm opaque tank. Therefore, the
fish could not be influenced by any outside disturbances. The tank had an open top and
was filled about half way with normoxic water. Water was then continuously pumped in
and out of the tank maintaining the half way depth. However, the water was pumped
slowly to keep the environment as calm as possible. Once a fish was in the tank, it was
given 5 minutes to acclimate to the new environment.
Mirror image stimulation (MIS) was the method used to assess aggression. It
has proven to be an effective technique for measuring fish behavior (Tinbergen, 1951).
A 10.16 cm high by 7.6 cm wide mirror was placed against the wall inside of the opaque
tank. The opposite side of the tank contained an artificial plant, providing the fish with
an opportunity to seek refuge. A video camera was attached above the tank, making
sure the entire area of the tank was in the viewing arena of the camera (Marks et al.,
2005).
Once the fish had been acclimated, it was recorded for four minutes. At the end
of the four minutes, the fish was removed and its wet mass was recorded. This
procedure was repeated for all the fish that survived the 150 days.
Once every fish was assayed, their respective video recordings were reviewed.
Each individual recording was reviewed from the one minute mark to the three minute
mark. Aggression was measured as the total time spent in contact with the mirror.
Avoidance was measured as total time spent pushed into the plastic plant. .
Statistics
I used a one way ANOVA to determine the effect of androgen treatment on
zebrafish behavior. I performed a natural log transformation of the raw data to achieve
the best normalization of residuals. Statistics were performed with SAS and the level of
significance was set at 0.05. Data presented are means +/- standard error.
Treatment
Aggression (s)
Avoidance (s)
Control (N=27)
0.59 +/- 0.16
2.26 +/- 0.33
Testosterone (N=28)
0.76 +/- 0.17
2.18 +/- 0.33
Estradiol (N=42)
0.61 +/- 0.14
2.87 +/- 0.29
Table 1. Means are presented with their standard error
Aggression
Testosterone treated fish had the highest aggression level followed by estradiol
treated and control fish (figure 1). However, ANOVA results indicated these differences
were not significant (P = 0.73).
Avoidance
Estradiol treated fish spent the most time hiding, followed by control and
testosterone treated fish (figure 2). However, ANOVA results indicated these differences
to be not significant (P = 0.21)
Aggression
0.9
0.8
0.7
Aggression (s)
0.6
0.5
0.4
0.3
0.2
0.1
0
Control (N=27)
Testosterone (N=28)
Estradiol (N=42)
Treatment
Figure 1. Mean times spent engaging in aggressive behavior for fish from all three
treatments. Error bars represent standard errors.
Avoidance
3.5
3
Avoidance (s)
2.5
2
1.5
1
0.5
0
Control (N=27)
Testosterone (N=28)
Estradiol (N=42)
Treatment
Figure 2. Mean times spent engaging in avoidance behavior for fish from all three
treatments. Error bars represent standard errors.
Discussion
The goal of this study was to determine if increased levels of testosterone or
estrogen in the embryonic and perinatal periods of zebrafish development influence
adult behavior. The results indicate that there is not enough evidence to claim early
androgen exposure effects adult behavior. There could be one or several explanations
for this. First, the concentrations of the treatment solutions could have been too low.
The 1.0 M concentrations used in this study may have been too low to up regulate
androgen or estrogen receptor activity in the developing fish. Another explanation is
that the fish were not exposed to their treatment for a long enough developmental
period. Perhaps exposing the fish through adolescence would have shown results
(Melloni & Ricci, 2010).
As stated in the introduction, the main male fish sex hormone is 11ketotestosterone. Our fish were treated with testosterone. Although both hormones
have been found to up-regulate the androgen receptor in fish (Lassiter & Linney, 2007),
the zebrafish receptor has been found to favor 11-ketotestosterone when it comes to
up-regulation (Hossain et al., 2008). Perhaps treating the fish with the favored hormone
would have shown a difference in the results.
The enzyme aromatase, which is responsible for the aromatization of
testosterone, may also be a key to explaining the results. Since aromatization has been
indirectly linked to aggression (Schlinger & Callard, 1990), decreased aromatization
could correlate with low levels of aggressive behavior. Low levels of aromatase will
lead to decreased aromatization and fish have been found to have low levels of
aromatase in the embryonic and perinatal periods (Lephart, 1996). The fish were
exposed to testosterone during this time period and without aromatase, the androgen
and estrogen receptors will not have been up-regulated. Another important finding is
that aromatase in fish is controlled by two genes, one in the gonads and one in the brain
(Tchoudakova et al., 2001). Mammals and birds, on the other hand, control aromatase
expression by a single gene (Conley & Hinshelwood, 2001). The studies linking the
enzyme with aggression were done with mammals.
A final explanation that has to be raised is that testosterone and estradiol are not
always linked to aggressive behaviors. This explanation has been the topic of a lot of
recent research. Aggression levels in humans administered testosterone have been
shown to be altered by social conditions (Eisenegger et al., 2010). If this finding is
shared between other animals, then zebrafish may only act aggressively under certain
social conditions. The design utilized in this study may have failed to account for
complex social interaction necessary to stimulate aggressive behavior. .
Even though our study did not yield statistically significant results, it still raised
important questions. Future research should address dosage, critical periods of
development, and the nature of social interactions as important factors. These studies
will yield important insights into the role hormones play in shaping animal behavior.
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