Ala 586 Asp mutation in androgen receptor disrupts transactivation function without affecting androgen binding  Singh Rajender, Ph.D., Nalini J. Gupta, Ph.D., Baidyanath Chakrabarty, M.D., Lalji Singh, Ph.D., Kumarasamy Thangaraj, Ph.D.  Fertility and Sterility  Volume 91, Issue 3, Pages 933.e23-933.e28 (March 2009) DOI: 10.1016/j.fertnstert.2008.10.041 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Pedigree structure of the family. (B) Phenotypic features of the index patient. (C) Histology of gonad sections showing well-differentiated seminiferous tubules with thick basement membrane (1) surrounded by fibroblasts (2). The interstitial space was filled with Leydig cells (3). Spermatogonium (4) could be spotted in the seminiferous tubules. The lumen of seminiferous tubules was filled with Sertoli cells (5). (D) Electropherogram showing the mutation in AR gene. The electropherogram for the patient is placed above the control. Fertility and Sterility 2009 91, 933.e23-933.e28DOI: (10.1016/j.fertnstert.2008.10.041) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Western blotting showing the normal expression of the AR protein. Lane M, protein weight marker; lane S, the patient sample; lane UT, untransfected sample (negative control); and lane WT, wild-type sample (positive control). Fertility and Sterility 2009 91, 933.e23-933.e28DOI: (10.1016/j.fertnstert.2008.10.041) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Ligand-binding assays for mutant clone. NSB-WT = nonspecific binding with the wild-type allele; NSB-MT = nonspecific binding with mutant allele. The data in the figure are an average of three independent experiments. (B) Transactivation assay for the mutant and wild-type clones. The downstream activity of the ligand–AR complex was measured by the extent of expression of a reporter gene. The data in the figure are an average of three independent experiments. The error bars are inferential showing standard error. Fertility and Sterility 2009 91, 933.e23-933.e28DOI: (10.1016/j.fertnstert.2008.10.041) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Extent of conservation of the mutated residues across mammalian species. The mutated amino acid is highlighted. (B) The location of the mutation in rat androgen receptor–DNA-binding domain crystal structure. The two zinc fingers are positioned relative to each other to favor binding of the hormone–receptor complex to target DNA sequences. The mutated amino acid residue (clear circle) is a part of the DNA recognition helix of the AR protein. Fertility and Sterility 2009 91, 933.e23-933.e28DOI: (10.1016/j.fertnstert.2008.10.041) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions