Transcript Androgen Receptor Localization in the Haplochromis burtoni

Androgen Receptor Localization in
the Haplochromis burtoni brain
Starring Haplochromis burtoni as the Territorial Male
The role of GnRH

Preoptic gonadotropin releasing hormone (GnRH-I)
neurons in the male teleost Haplochromis burtoni
have been shown to integrate social cues and
neuroendocrine information.

GnRH-I upregulates the release of pituitary
gonadotropins, which in turn, upregulates the
secretion of androgens from the testes. Gonadal
androgens then complete the loop by negatively
regulating the release of preoptic GnRH-I through
interaction with androgen receptors (location unknown).
Model for GnRH-I regulation
Social Setpoint
+/-
Preoptic
Area
GnRH-I
+
Pituitary
-Gonadotropins
Androgens
Gonads
+
Neuroanatomy
Androgen Receptor Domains
DNA
Binding Hinge
Transactivation
A/B
White 57
17 aa
C
White 275
19 aa
D
Ligand Binding
E(F)
Hypothesis
Since androgens may regulate the abundance of GnRH-I
mRNA within the preoptic area, at least one of the
following hypotheses must be correct:



Androgens activate receptors within preoptic
neurons coexpressing GnRH-I
AR are in neurons that do not coexpress GnRH-I
but are in the same area
AR are located in neurons in another area which
then project to GnRH-I containing neurons
*It’s important to note that these hypotheses are not
mutually exclusive
Methodology
I. General LocalizationThe expression of AR will be assessed using
immunocytochemistry. This sensitive technique will allow
observation of where the AR are located in the brain.
Androgen Receptor
Chicken Polyclonal Primary Ab
Biotinylated polyclonal
goat anti-chicken Secondary Ab
Avidin D- fluorescein (FITC)
IV. Analysis• Stained sections observed using a fluorescent
microscope
• Images captured with a digital camera and
downloaded onto a computer
• Localization of AR determined first by comparing
tissue to known neuroanatomical regions, then
through comparison with slides themselves by
staining cells with GnRH-I antibodies
Experiment 1
Conditions
Results
Used different dilutions (1:50, 1:100,
1:200, 1:500. 1:1000, 1:5000) of both
primary Ab (White 57-from animal
402, and White 275-from animal
401). Also had one slide with only
blocking buffer (no primary Ab).
1:200 dilution of secondary Ab
(Biotinylated Anti-chicken IgG) and
of Avidin covalently coupled to
FITC used for all stainings.
As expected smaller dilutions
looked brighter when viewing
under fluorescence. Determined
1:500 dilution of primary Ab looked
best to use for future experiments.
No noticeable difference in binding
affinity between the different
primary Abs. Lots of background.
Experiment 2
Conditions
1:500 dilutions of both primary Ab.,
over the weekend incubation.
This time we also added boiling 10
mmol Na citrate (ph=6) to some
slides after PBS washing step in
order to try to unmask/expose
androgen receptor (the antigen)
more.
Results
Longer incubation time did not
appear to improve staining.
Attempt at unmasking did not
result in better staining, still
observing a large noise to signal
ratio. Hard to see any distinct
labeling due to large amount of
background observed.
Experiment 3
Conditions
Results
1:500 dilution of both primary Ab
used. But this time used tissue that
was not fixed in paraformaldehyde
but instead frozen on dry ice. This
was to see if maybe we were
overfixing the tissue  thereby
making it harder for the Ab to bind
to the antigen, resulting in no
distinct labeling
This time also stained with DAPI to
be able to see cell nuclei.
Not much difference from previous
stainings. Still getting a lot of
background, and still hard to
observe any distinct labeling.
So, what’s next?
For the next staining, we will be using an
antibody isolated from the yolk of the chicken
eggs. This will let us observe if the yolk (IgY)
antibody has a greater (more selective) binding
affinity than the serum primary Ab that we have
been using thus far.
Let’s keep our fingers crossed……
Future Directions
I. ColocalizationA double labeling protocol will be used to determine
whether same cells co-express GnRH- I and AR
proteins. This will involve using two fluorophores, one
green in color (fluorescein) and the second red in color
(rhodamine). Where these two fluorophores are
colocalized yellow staining will be observed.
II. Social status affects on AR localizationMales of different social status: T, NT, individuals
transitioning into T (or into NT) status will be used to
study if changes in social status affect AR localization