Expression of novel androgen receptors in three GnRH neuron subtypes in the cichlid brain

In teleosts, GnRH1 neurons stand at the apex of the Hypothalamo-Pituitary-Gonadal (HPG) axis, which is responsible for the production of sex steroids by the gonads (notably, androgens). To exert their actions, androgens need to bind to their specific receptors, called androgen receptors (ARs). Due to a teleost-specific whole genome duplication, A. burtoni possess two AR paralogs (ARα and ARβ) that are encoded by two different genes, ar1 and ar2, respectively. In A. burtoni, males stratify along dominance hierarchies, in which an individuals' social status determines its physiology and behavior. GnRH1 neurons have been strongly linked with dominance and circulating androgen levels. Similarly, GnRH3 neurons are implicated in the display of male specific behaviors. Some studies have shown that these GnRH neurons are responsive to fluctuations in circulating androgens levels, suggesting a link between GnRH neurons and ARs. While female A. burtoni do not naturally form a social hierarchy, their reproductive state is positively correlated to androgen levels and GnRH1 neuron size. Although there are reports related to the expression of ar genes in GnRH neurons in cichlid species, the expression of each ar gene remains inconclusive due to technical limitations. Here, we used immunohistochemistry, in situ hybridization chain reaction (HCR), and spatial transcriptomics to investigate ar1 and ar2 expression specifically in GnRH neurons. We find that all GnRH1 neurons intensely express ar1 but only a few of them express ar2, suggesting the presence of genetically-distinct GnRH1 subtypes. Very few ar1 and ar2 transcripts were found in GnRH2 neurons. GnRH3 neurons were found to express both ar genes. The presence of distinct ar genes within GnRH neuron subtypes, most clearly observed for GnRH1 neurons, suggests differential control of these neurons by androgenic signaling. These findings provide valuable insight for future studies aimed at disentangling the androgenic control of GnRH neuron plasticity and reproductive plasticity across teleosts.