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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 ci trate (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