2. Androgen Receptor Localization in the Haplochromis burtoni brain Starring Haplochromis burtoni as the Territorial Male

3. The role of GnRH n Preoptic gonadotropin releasing hormone (GnRH-I) neurons in the male teleost Haplochromis burtoni have been shown to integrate social cues and neuroendocrine information. n 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).

5. Model for GnRH-I regulation Preoptic Area Area Social Setpoint +/- GnRH-I + Pituitary Gonads Androgens Gonadotropins + --

6. Neuroanatomy

7. Androgen Receptor Domains Transactivation DNABinding Hinge Ligand Binding A/BCDE(F) White 57 17 aa White 275 19 aa

8. Hypothesis n Androgens activate receptors within preoptic neurons coexpressing GnRH-I n AR are in neurons that do not coexpress GnRH-I but are in the same area n 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

9. Methodology General 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)

10. 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.

11. Experiment 1

12. Experiment 2

13. Experiment 3

14. 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……

15. 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