Validation of custom antibodies against androgen receptors alpha and beta in Astatotilapia burtoni

Astatotilapia burtoni are unique in that they have two paralogs of androgen receptors (AR), ARalpha and ARbeta. Gonadotropin releasing hormone (GnRH) neurons regulate reproductive hormone levels and modulate physiology and behavior. This makes their study important to fertility and hormone imbalance disorders linked to androgens (male sex hormones such as testosterone that are present in females too) and GnRH neurons, such as polycystic ovarian syndrome. Whilst previous research has shown that androgen signaling affects the morphology and function of GnRH neurons, it is unclear how they exert these effects. To uncover these mechanisms, understanding the spatial relationship of the two androgen receptors and GnRH-1 neurons is imperative. Custom antibodies made against AR alpha or AR beta (Aves lab) were used to label ARalpha and ARbeta, at 1:500 or 1:1000 concentration. We found that the 1:500 concentration optimized the visibility of cells underneath a fluorescence microscope. Preliminary data showed nuclear ARalpha in the ventral pre-optic area (POA) in a location similar to that of GnRH-neurons, suggesting there may be colocalization of ARalpha and GnRH neurons in future double labels. ARbeta in the tectum is reminiscent of neuropil structures or hypothalamic projections. However, whilst it has been previously assumed that ARbeta is a nuclear protein, the irregular and non-circular shape of the stain suggests otherwise. Further research is required to assess the exact location of AR protein and the brain structure they reside.