Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand.
A microassay for the androgen receptor was developed to investigate the cellular distribution of receptor in freshly isolated testicular cell types. The microassay uses an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is rapid and irreversible, which permits the development of a highly sensitive assay. The androgen receptor microassay is completed within 4 h and detects receptor in as little as 0.5 micrograms cellular protein. There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, including androgen-binding protein. Androgen receptor was found in cellular sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Although the relative distribution of receptor was similar to that obtained using a traditional equilibrium binding assay, the levels of receptor were significantly higher using the microassay. The androgen receptor microassay was subsequently used to investigate the receptor in isolated testicular cell types. Androgen receptor was detected in freshly isolated peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a mixed population of germ cells. Hormones were not found to influence androgen receptor levels in cultured peritubular cells or Sertoli cells. Electrophoretic analysis of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa form of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The size of the androgen receptor species detected in the rat testicular cell types was slightly smaller than the 56-kDa protein detected in a human fibroblast cell line. The current study demonstrates the utility of the microassay and affinity ligand to investigate androgen receptor biology. Data indicate that androgen receptors are present in several testicular cell types and suggest that the control of testicular function by androgens probably involves actions on multiple cell types.
Anthony
C T
CT
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Kovacs
W J
WJ
Skinner
M K
MK
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D009928:Organ Specificity; D051381:Rats; D011944:Receptors, Androgen / Q000032:analysis* / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism; D012867:Skin / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000378:metabolism*
D000345:Affinity Labels; D011944:Receptors, Androgen; D013196:Dihydrotestosterone; C023562:dihydrotestosterone 17-bromoacetate