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

PreopticPreoptic AreaArea

Social Setpoint Social Setpoint +/-+/-

GnRH-I GnRH-I ++PituitaryPituitary

GonadsGonadsAndrogensAndrogens

GonadotropinsGonadotropins

++

----

Neuroanatomy

Androgen Receptor Domains

TransactivationTransactivationDNADNA

BindingBinding HingeHinge Ligand BindingLigand Binding

A/BA/B CC DD E(F)E(F)

White 5717 aa

White 27519 aa

Hypothesis

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

Since androgens may regulate the abundance of GnRH-I mRNA within the preoptic area, at least one of the following hypotheses must be correct:

*It’s important to note that these hypotheses are not mutually exclusive

MethodologyGeneral Localization-The expression of AR will be assessed using immunocytochemistry. This sensitive technique will allow observation of where the AR are located in the brain.

I.

Androgen Receptor

Chicken Polyclonal Primary Ab

Biotinylated polyclonal goat anti-chicken Secondary Ab

Avidin D- fluorescein (FITC)

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

IV.

Experiment 1

Conditions Results

Used different dilutions (1:50, 1:100,1:200, 1:500. 1:1000, 1:5000) of bothprimary Ab (White 57-from animal402, and White 275-from animal401). Also had one slide with onlyblocking buffer (no primary Ab).1:200 dilution of secondary Ab(Biotinylated Anti-chicken IgG) andof Avidin covalently coupled toFITC used for all stainings.

As expected smaller dilutionslooked brighter when viewingunder fluorescence. Determined1:500 dilution of primary Ab lookedbest to use for future experiments.No noticeable difference in bindingaffinity between the differentprimary Abs. Lots of background.

Experiment 2

Conditions Results

1:500 dilutions of both primary Ab.,over the weekend incubation.This time we also added boiling 10mmol Na citrate (ph=6) to someslides after PBS washing step inorder to try to unmask/ exposeandrogen receptor (the antigen)more.

Longer incubation time did notappear to improve staining.Attempt at unmasking did notresult in better staining, stillobserving a large noise to signalratio. Hard to see any distinctlabeling due to large amount ofbackground observed.

Experiment 3

Conditions Results

1:500 dilution of both primary Abused. But this time used tissue thatwas not fixed in paraformaldehydebut instead frozen on dry ice. Thiswas to see if maybe we wereoverfixing the tissue ‡ therebymaking it harder for the Ab to bindto the antigen, resulting in nodistinct labelingThis time also stained with DAPI tobe able to see cell nuclei.

Not much difference from previousstainings. Still getting a lot ofbackground, and still hard toobserve 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……

Colocalization-A 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.

I.

Social status affects on AR localization-Males 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

II.

Future Directions