Virilization of the female spotted hyena cannot be explained by alterations in the amino acid sequence of the androgen receptor (AR)

The external genitalia of the female spotted hyena are male in character, consistent with virilization by androgens during embryogenesis that results in the fusion of the vaginal labia to form a pseudo scrotum and enlargement of the clitoris to form a phallus. Explanations advanced to account for these anatomic differences have centered on the production or metabolism of androgens in utero or on abnormalities of the androgen receptor (such as a constitutively active AR). The structure of the spotted hyena AR was examined at the level of genomic DNA and cDNA. Southern analysis detected two Eco RI endonuclease cleavage fragments (4.4 and 4.7 kb) that encode the bulk of the AR hormone-binding domain. Isolation of the smaller fragment from a size fractionated genomic library revealed that it contained exons 6, 7 and 8. The remaining portions of the coding sequence were cloned by RT-PCR and RACE analyses. The spotted hyena cDNA sequence predicts protein 912 amino acids in length, which is most closely related to the sequence of the dog AR. Although a number of differences in the predicted amino acid sequence are identified, particularly within the amino terminus, only single amino acid substitutions are present in the DNA- and ligand-binding domains compared to the human AR. In transfection assays, the spotted hyena AR does not exhibit constitutive activity and responds normally to a range of androgenic and non-androgenic ligands. These findings suggest that the structural changes in the AR do not account for the abnormal virilization in the female spotted hyena. These results serve to focus attention on processes proximal (an abnormality of hormone formation in situ) or distal (activation by other mechanisms of processes normally regulated by androgen) to the AR as the cause of the virilization.