Endocrine Abstracts

The importance of the androgen receptor (AR) in the development and maintenance of the male phenotype is demonstrated by patients with androgen insensitivity syndrome (AIS). These patients carry germline mutations in the AR and display a mild to complete intersex phenotype. The AR belongs to the subfamily of steroid receptors, which need to form homodimers to execute their role as transcription factors. For estrogen receptors, dimerization via the DNA-binding domains as well as via the ligand-binding domains (LBDs) is well documented. For the AR, however, LBD dimerization has only recently been identified by means of a crystal structure of AR LBDs. In In vitro assays, AR LBD dimerization was agonist-dependent and inhibited by androgen antagonists. We also showed that some AIS mutations in the AR LBD indeed disrupt this dimerization interface. A clear example is the W752R mutation, found in two siblings with AIS but information about the severity of their phenotype is lacking¹. In vitro, this mutation was still able to induce androgen reporter genes albeit at higher hormone concentrations.

To study the physiological relevance of AR LBD dimerization, we introduced the corresponding mutation (W731R) by CRISPR/Cas9 into the germ line of mice. The thus generated ARLmon/Y males have an external female phenotype with small, cryptorchid testes, yet high circulating levels of testosterone, androstenedione and LH. Prostate, seminal vesicles and epididymis did not develop. Despite the elevated testosterone levels, the AR^Lmon/Y mice have a severe bone phenotype, similar to the bone phenotype of complete AR knockout (ARKO) mice. Because the AR^Lmon/Y mice have high circulating levels of testosterone, which can still function as prohormone for estradiol, this observation suggests a direct effect of the AR on bone homeostasis. Heterozygous AR^Lmon/X female mice had normal fertility, number of litters and pups per litter.

Transcriptomic and immunohistological analysis of the AR^Lmon/Y testes showed hyperplasia of the Leydig cells, presence of Sertoli cells and residual spermatogenesis. The AR responses where completely absent in Leydig and Sertoli cells as well as in other androgen-responsive organs. Indeed, castration experiments with supraphysiological testosterone replacement confirmed absence of androgen response in AR^Lmon/Y kidneys while AR regulation was observed in wild type kidneys. Furthermore, analysis of the steroidogenic pathway revealed that the expression of Hsd17b3, which is responsible for the conversion of androstenedione into testosterone, is low in AR^Lmon/Y testis. Reporter assays demonstrated that the transcription of this gene is regulated by the AR itself.

In conclusion, the AR^Lmon/Y mouse model reveals the physiological importance of LBD dimerization of the AR. This is the first report of a mouse model with a disrupted LBD dimerization of a steroid receptor.

Reference: 1. Boehmer AL, et al. (2001) Genotype versus phenotype in families with androgen insensitivity syndrome. J Clin Endocrinol Metab 86(9):4151–4160.